Adeno-associated virus formulations

ABSTRACT

Adeno-associated liquid and lyophilized pharmaceutical compositions are provided herein. In exemplary aspects, the pharmaceutical compositions comprise about 5 mM to about 25 mM L-histidine, about 0 mM to about 150 mM sodium chloride, about 0.001% (w/v) to about 0.01% (w/v) polysorbate 80 (PS80), and about 1% to about 10% (w/v) sucrose, trehalose, or combination thereof to AAV. In exemplary aspects, the pharmaceutical compositions further comprise glycine or mannitol. Methods of preparing a pharmaceutical composition comprising AAV, methods of treating a bleeding disorder in a subject, and methods of storing AAV compositions are also provided.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/417,750 filed Nov. 4, 2016, which is hereby incorporated byreference in its entirety.

BACKGROUND

Adeno-associated virus (AAV) is a small, non-enveloped virus thatpackages a linear single-stranded DNA genome. AAV belongs to the familyParvoviridae and the genus Dependovirus, since productive infection byAAV occurs only in the presence of a helper virus, such as, for example,adenovirus or herpes virus. Even in the absence of a helper virus, AAV(serotype 2) can achieve latency by integrating into chromosome 19q13.4of a host human genome. It is the only mammalian DNA virus known to becapable of site-specific integration (Daya and Berns, ClinicalMicrobiology Reviews, pages 583-593 (2008)).

For AAV to be safely used in the clinic, AAV has been geneticallymodified at several locations within its genome. For example, the Repgene, which is required for viral replication, and the element requiredfor site-specific integration have been eliminated from the AAV genomein many viral vectors. This recombinant AAV (rAAV), exists in anextrachromosomal state and have very low integration efficiency into thegenomic DNA. The possibility of rAAV inducing random mutagenesis in ahost cell is thus reduced, if not eliminated altogether. Because ofthese properties and the lack of pathogenicity, rAAV has shown greatpromise as a gene therapy vector in multiple aspects of pre-clinical andclinical applications. New serotypes and self-complementary vectors arebeing tested in the clinic. Alongside these ongoing vector developments,continued effort has focused on scalable manufacturing processes thatcan efficiently generate high titer quantities of rAAV vectors with highpurity and potency.

AAV research also has focused on AAV formulations intended for humanadministration. It is understood that such AAV formulations should benot only safe, sterile, and of good manufacturing practice (GMP) grade,these formulations should also exhibit and promote the long-termstability of the AAV, minimizing loss of AAV potency during themanufacture, packaging, and storage processes. The formulations shouldfurthermore prevent adsorption to the surfaces of the containers inwhich the AAV are packaged and stored and of the machinery used duringmanufacture. Though the efforts to design such AAV formulations havebeen great, there still remains a need for improved AAV formulations.

SUMMARY

The present disclosure provides formulations compatible for humanadministration which address the unmet needs described above.Advantageously, in some embodiments, the formulations are suitable forlong-term storage of AAV, minimizing loss of AAV potency, andadvantageously prevent visible particle formation and prevent adsorptionto the surfaces of the containers in which the AAV are packaged andstored and of the machinery used during manufacture. In exemplaryaspects, the pharmaceutical composition of the present disclosurecomprises: adeno-associated virus (AAV) and about 5 mM to about 25 mM ofa buffering agent, about 0 mM to about 150 mM of a pharmaceuticallyacceptable salt, about 0.001% (w/v) to about 0.01% (w/v) of a non-ionicsurfactant, and about 1% (w/v) to about 10% (w/v) of a sugar or sugaralcohol. In certain embodiments, the formulation comprises about 50 mMto about 150 mM of a pharmaceutically acceptable salt. In certainembodiments, the formulation comprises about 0 mM to less than about 100mM of a pharmaceutically acceptable salt. In certain embodiments, theformulation comprises about 30 mM to less than about 100 mM of apharmaceutically acceptable salt. In certain embodiments, theformulation comprises about 20 mM to about 40 mM of a pharmaceuticallyacceptable salt.

In exemplary aspects, the pharmaceutical composition of the presentdisclosure comprises: AAV (e.g., AAV8), about 5 mM to about 25 mML-histidine, about 0 mM to about 150 mM sodium chloride, about 0.001%(w/v) to about 0.01% (w/v) polysorbate 80 (PS80), and about 1% to about10% (w/v) sucrose, trehalose, mannitol, or a combination thereof. Incertain embodiments, the formulation comprises about 50 mM to about 150mM sodium chloride. In certain embodiments, the formulation comprisesabout 0 mM to less than about 100 mM sodium chloride. In certainembodiments, the formulation comprises about 30 mM to less than about100 mM sodium chloride. In certain embodiments, the formulationcomprises about 20 mM to about 40 mM sodium chloride.

In some embodiments, the invention provides a liquid formulation. Incertain embodiments, the formulation is lyophilized from a liquidformulation.

In certain aspects, the lyophilized pharmaceutical composition of thepresent disclosure is lyophilized from a liquid formulation comprising:AAV (e.g., AAV8), about 5 mM to about 25 mM L-histidine, about 0 mM toabout 150 mM sodium chloride, about 0.001% (w/v) to about 0.01% (w/v)polysorbate 80 (PS80), about 30 mM to about 70 mM glycine, and about 1%to about 10% (w/v) sucrose, trehalose, mannitol, or a combinationthereof. In certain embodiments, the formulation comprises about 50 mMto about 150 mM sodium chloride. In certain embodiments, the formulationcomprises about 0 mM to less than about 100 mM sodium chloride. Incertain embodiments, the formulation comprises about 30 mM to less thanabout 100 mM sodium chloride. In certain embodiments, the formulationcomprises about 20 mM to about 40 mM sodium chloride.

Methods of preparing a pharmaceutical composition comprising AAV arefurther provided herein. In exemplary aspects, the method comprisescombining about 5 mM to about 25 mM L-histidine, about 0 mM to about 150mM sodium chloride, about 0.001% (w/v) to about 0.01% (w/v) polysorbate80 (PS80), about 1% (w/v) to about 10% (w/v) sucrose, trehalose,mannitol, or a combination thereof and AAV, thereby obtaining apharmaceutical composition comprising AAV. In certain embodiments, theformulation comprises about 50 mM to about 150 mM sodium chloride. Incertain embodiments, the formulation comprises about 0 mM to less thanabout 100 mM sodium chloride. In certain embodiments, the formulationcomprises about 30 mM to less than about 100 mM sodium chloride. Incertain embodiments, the formulation comprises about 20 mM to about 40mM sodium chloride.

Methods of treating a subject for a disorder treatable by gene therapyare provided by the present disclosure. In exemplary aspects, the methodcomprises administering to the subject a pharmaceutical composition asdescribed herein in an amount effective to treat the disorder.

Methods of storing a composition comprising AAV are moreover providedherein. In exemplary aspects, the method comprises combining about 5 mMto about 25 mM L-histidine, about 0 mM to about 150 mM sodium chloride,about 0.001% (w/v) to about 0.01% (w/v) polysorbate 80 (PS80), about 1%(w/v) to about 10% (w/v) sucrose, trehalose, mannitol, or a combinationthereof and AAV. In certain embodiments, the formulation comprises about50 mM to about 150 mM sodium chloride. In certain embodiments, theformulation comprises about 0 mM to less than about 100 mM sodiumchloride. In certain embodiments, the formulation comprises about 30 mMto less than about 100 mM sodium chloride. In certain embodiments, theformulation comprises about 20 mM to about 40 mM sodium chloride.

In certain embodiments, the pharmaceutical composition of the presentdisclosure comprises: AAV and about 20 mM L-histidine, about 70 mMsodium chloride, about 0.005% (w/v) polysorbate 80 (PS80), and about 5%(w/v) sucrose. In some embodiments, the invention provides a liquidformulation. In certain embodiments, the formulation is lyophilized froma liquid formulation.

In certain embodiments, the pharmaceutical composition of the presentdisclosure comprises: AAV and about 20 mM L-histidine, about 60 mMsodium chloride, about 0.005% (w/v) polysorbate 80 (PS80), about 35 mMtrehalose, and about 110 mM mannitol. In some embodiments, the inventionprovides a liquid formulation. In certain embodiments, the formulationis lyophilized from a liquid formulation.

In certain embodiments, the pharmaceutical composition of the presentdisclosure comprises: AAV and about 10 mM L-histidine, about 100 mMsodium chloride, about 0.005% (w/v) polysorbate 80 (PS80), about 5%(w/v) trehalose, and about 50 mM glycine. In some embodiments, theinvention provides a liquid formulation. In certain embodiments, theformulation is lyophilized from a liquid formulation.

In certain embodiments, the pharmaceutical composition of the presentdisclosure comprises: AAV and about 10 mM L-histidine, about 80 mMsodium chloride, about 0.005% (w/v) polysorbate 80 (PS80), about 5%(w/v) trehalose, and about 50 mM glycine. In some embodiments, theinvention provides a liquid formulation. In certain embodiments, theformulation is lyophilized from a liquid formulation.

In certain embodiments, the pharmaceutical composition of the presentdisclosure comprises: AAV and about 10 mM L-histidine, about 70 mMsodium chloride, about 0.005% (w/v) polysorbate 80 (PS80), about 5%(w/v) trehalose, and about 50 mM glycine. In some embodiments, theinvention provides a liquid formulation. In certain embodiments, theformulation is lyophilized from a liquid formulation.

In certain embodiments, the pharmaceutical composition of the presentdisclosure comprises: AAV and about 10 mM L-histidine, about 60 mMsodium chloride, about 0.005% (w/v) polysorbate 80 (PS80), about 5%(w/v) trehalose, and about 50 mM glycine. In some embodiments, theinvention provides a liquid formulation. In certain embodiments, theformulation is lyophilized from a liquid formulation.

In certain embodiments, the pharmaceutical composition of the presentdisclosure comprises: AAV and about 10 mM L-histidine, about 50 mMsodium chloride, about 0.005% (w/v) polysorbate 80 (PS80), about 5%(w/v) trehalose, and about 50 mM glycine. In some embodiments, theinvention provides a liquid formulation. In certain embodiments, theformulation is lyophilized from a liquid formulation.

In certain embodiments, the pharmaceutical composition of the presentdisclosure comprises: AAV and about 10 mM L-histidine, about 40 mMsodium chloride, about 0.005% (w/v) polysorbate 80 (PS80), about 5%(w/v) trehalose, and about 50 mM glycine. In some embodiments, theinvention provides a liquid formulation. In certain embodiments, theformulation is lyophilized from a liquid formulation.

In certain embodiments, the pharmaceutical composition of the presentdisclosure comprises: AAV and about 10 mM L-histidine, about 30 mMsodium chloride, about 0.005% (w/v) polysorbate 80 (PS80), about 5%(w/v) trehalose, and about 50 mM glycine. In some embodiments, theinvention provides a liquid formulation. In certain embodiments, theformulation is lyophilized from a liquid formulation.

In certain embodiments, the pharmaceutical composition of the presentdisclosure comprises: AAV and about 10 mM L-histidine, about 20 mMsodium chloride, about 0.005% (w/v) polysorbate 80 (PS80), about 5%(w/v) trehalose, and about 50 mM glycine. In some embodiments, theinvention provides a liquid formulation. In certain embodiments, theformulation is lyophilized from a liquid formulation.

In certain embodiments, the pharmaceutical composition of the presentdisclosure comprises: AAV and about 10 mM L-histidine, about 10 mMsodium chloride, about 0.005% (w/v) polysorbate 80 (PS80), about 5%(w/v) trehalose, and about 50 mM glycine. In some embodiments, theinvention provides a liquid formulation. In certain embodiments, theformulation is lyophilized from a liquid formulation.

In certain embodiments, the pharmaceutical composition of the presentdisclosure comprises: AAV and about 10 mM L-histidine, about 0 mM sodiumchloride, about 0.005% (w/v) polysorbate 80 (PS80), about 5% (w/v)trehalose, and about 50 mM glycine. In some embodiments, the inventionprovides a liquid formulation. In certain embodiments, the formulationis lyophilized from a liquid formulation.

Generally, the AAV formulations provided herein are suitable forpharmaceutical administration. In certain embodiments, the AAV is AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In certainembodiments, the AAV is AAV 2, AAV 5, AAV 8, or AAV 9. In certainembodiments, the AAV is AAV8.

DETAILED DESCRIPTION

The present disclosure provides formulations, e.g., pharmaceuticalcompositions, compatible for human or veterinarian administration whichalso are suitable for long-term storage of AAV and minimizing loss ofAAV potency. The formulations provided herein are advantageous, becausethe formulations prevent visible particle formation and also preventadsorption to the surfaces of the containers in which the AAV arepackaged and stored, and prevent adsorption to the machinery used duringmanufacture. In certain embodiments, the pharmaceutical compositionsprovided herein retain significant AAV activity when stored for extendedperiods of time. In certain embodiments, the pharmaceutical compositionsprovided herein reduce or retard degradation and/or aggregation.

In certain embodiments, the present invention provides formulations ofAAV comprising a therapeutically effective amount or dose of an AAV, asub-physiological to physiological concentration of a pharmaceuticallyacceptable salt, a stabilizing concentration of one or more sugarsand/or sugar alcohols, a non-ionic surfactant, one or more bufferingagents providing a neutral pH to the formulation, and optionally anamino acid to aid in stability and recovery of the AAV duringpurification and/or processing. Generally, the AAV formulations providedherein are suitable for pharmaceutical administration. In certainembodiments, the AAV is AAV8.

In certain embodiments, the composition that contains the AAV is adehydrated composition. As used herein, a dehydrated composition is acomposition that includes water in a low amount, such as 25% or less, or20% or less, or 15% or less, or 10% or less, or 9% or less, or 8% orless, or 7% or less, or 6% or less, or 5% or less, or 4% or less, or 3%or less, or 2% or less, or 1% or less water as measured by Karl Fischer(KF) titration. In certain embodiments, a dehydrated composition has 3%or less water as measured by Karl Fischer titration. In certainembodiments, a dehydrated composition has 2% or less water as measuredby Karl Fischer titration. In certain embodiments, a dehydratedcomposition has 1% or less water as measured by Karl Fischer titration.In certain embodiments, a dehydrated composition has 0.9% or less wateras measured by Karl Fischer titration. In certain embodiments, adehydrated composition has 0.8% or less water as measured by KarlFischer titration. In certain embodiments, a dehydrated composition has0.7% or less water as measured by Karl Fischer titration. In certainembodiments, a dehydrated composition has 0.6% or less water as measuredby Karl Fischer titration. In certain embodiments, a dehydratedcomposition has 0.5% or less water as measured by Karl Fischertitration. In certain embodiments, a dehydrated composition has 0.4% orless water as measured by Karl Fischer titration. In certainembodiments, a dehydrated composition has 0.3% or less water as measuredby Karl Fischer titration. In certain embodiments, a dehydratedcomposition has 0.2% or less water as measured by Karl Fischertitration. Any convenient protocol may be used to produce a dehydratedcomposition, such as increasing the temperature of the composition(e.g., heating), reducing the pressure, lyophilization (also known asfreeze-drying), and the like, and combinations thereof. Other methodsfor determining the moisture content of the composition includes, but isnot limited to, loss of drying (LOD) (measures the amount of water andvolatile matters in a sample when the sample is dried under specificconditions), electrolytic sensors (e.g., using a P2O5 sensor),Piezoelectric sorption, oxide sensors, aluminum oxide sensors,absorption spectroscopy, and near infrared (NIR).

Further, it was found that reducing the salt concentration in thelyophilized formulation reduced the residual moisture in the lyocake andassists in the formation of an improved lyocake.

Due to the low water content of a lyophilized composition as describedabove, the lyophilized composition may be in the form of a solid. Insome cases, the solid lyophilized composition is a powder. In somecases, a lyophilized composition may facilitate storage of thecomposition for an extended period of time (e.g., as compared to aliquid formulation of the same composition). For instance, a lyophilizedcomposition may be a storage stable composition (e.g., a lyophilizedstorage stable composition), where the composition is substantiallystable for an extended period of time. By “stable” or “storage stable”or “substantially stable” is meant a composition that does notsignificantly degrade and/or lose activity over an extended period oftime. For example, a storage stable composition may not have significantimpurities due to degradation of the composition over an extended periodof time, such as 10% or less impurities, or 9% or less, or 8% or less,or 7% or less, or 6% or less, or 5% or less, or 4% or less, or 3% orless, or 2% or less, or 1% or less degradation products over an extendedperiod of time. In certain instances, a storage stable composition has5% or less impurities over an extended period of time. In some cases, astorage stable composition substantially retains its activity over anextended period of time, such as retains 100% of its activity, or 99% ormore, or 98% or more, or 97% or more, or 96% or more, or 95% or more, or94% or more, or 93% or more, or 92% or more, or 91% or more, or 90% ormore, or 85% or more, or 80% or more, or 75% or more of its activityover an extended period of time. For example, a storage stablecomposition may retain 90% or more of its activity over an extendedperiod of time. In some cases, a storage stable composition retains 95%or more of its activity over an extended period of time. An extendedperiod of time is a period of time such as 1 week or more, or 2 weeks ormore, or 3 weeks or more, or 1 month or more, or 2 months or more, or 3months or more, or 4 months or more, or 6 months or more, or 9 months ormore, or 1 year or more, or 1.5 years (e.g., 18 months) or more, or 2years or more, or 2.5 years (e.g., 30 months) or more, or 3 years ormore, or 3.5 years (e.g., 42 months) or more, or 4 years or more, or 4.5years (e.g., 54 months) or more, or 5 years or more. For instance, anextended period of time may be 6 months or more. In some cases, anextended period of time is 9 months or more. In some cases, an extendedperiod of time is 1 year (e.g., 12 months) or more. In some cases, anextended period of time is 1.5 years (e.g., 18 months) or more. In somecases, an extended period of time is 2 years (e.g., 24 months) or more.In some embodiments, a storage stable composition is substantiallystable for an extended period of time at ambient temperature, such as atemperature of 20 to 40° C., or 25 to 35° C., or 25 to 30° C. In someinstances, a storage stable composition is substantially stable for anextended period of time at a temperature less than ambient temperature,such as a temperature of 0 to 20° C., or 0 to 15° C., or 0 to 10° C., or2 to 8° C.

Definitions

As used herein, the term “AAV” refers to adeno-associated virus in bothnaturally occurring and recombinant forms (rAAV), and encompasses mutantforms of AAV. The term AAV further includes, but is not limited to, AAVtype 1, AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV type 6, AAVtype 7, AAV type 8, AAV type 9, AAV type 10, avian AAV, bovine AAV,canine AAV, equine AAV, ovine AAV, primate AAV, and non-primate AAV. Incertain embodiments, the AAV is AAV8.

The phrase “pharmaceutically acceptable,” as used in connection withcompositions described herein, refers to molecular entities and otheringredients of such compositions that are physiologically tolerable anddo not typically produce untoward reactions when administered to asubject. Preferably, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in mammals, and more particularly in humans. A“pharmaceutically acceptable salt” is a salt that can be formulated intoa compound or conjugate for pharmaceutical use including, e.g., metalsalts (sodium, potassium, magnesium, calcium, etc.) and salts of ammoniaor organic amines that is safe for administration to a subject (e.g., ahuman) in a drug formulation (see, for example, Berge, et al.“Pharmaceutical Salts,” J. Pharm. Sci. 1977; 66:1, which is incorporatedherein by reference in its entirety and for all purposes). Suitable“pharmaceutically acceptable salts” include, but are not limited to,metal salts such as sodium, potassium and cesium salts; alkaline earthmetal salts such as calcium and magnesium salts; organic amine saltssuch as triethylamine, guanidine and N-substituted guanidine salts,acetamidine and N-substituted acetamidine, pyridine, picoline,ethanolamine, triethanolamine, dicyclohexylamine, andN,N′-dibenzylethylenediamine salts. “Pharmaceutically acceptable salts”(of basic nitrogen centers) include, but are not limited to inorganicacid salts such as the hydrochloride, hydrobromide, sulfate, phosphate;organic acid salts such as trifluoroacetate and maleate salts;sulfonates such as methanesulfonate, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, camphor sulfonate and naphthalenesulfonate; aminoacid salts such as arginate, alaninate, asparaginate and glutamate; andcarbohydrate salts such as gluconate and galacturonate. The selectionand use of pharmaceutically acceptable salts is well known in the art,for example, see Stahl and Wermuth, Pharmaceutical Salts: Properties,Selection, and Use, 2nd Revised edition, Wiley, Hoboken, N.J., which isincorporated herein by reference in its entirety and for all purposes.Non-limiting examples of pharmaceutically acceptable salts include,without limitation, sodium salts, ammonium salts, potassium salts (e.g,sodium, ammonium, and potassium chloride; sodium, ammonium, andpotassium acetate; sodium, ammonium, and potassium citrate; sodium,ammonium, and potassium phosphate; sodium, ammonium, and potassiumfluoride; sodium, ammonium, and potassium bromide; and sodium, ammonium,and potassium iodide).

As used herein, a “physiological concentration” of salt refers to a saltconcentration of between about 100 mM and about 200 mM of apharmaceutically acceptable salt.

As used herein, a “sub-physiological concentration” of salt refers to asalt concentration of less than about 100 mM of a pharmaceuticallyacceptable salt. In certain embodiments, a sub-physiologicalconcentration of salt is less than about 80 mM of a pharmaceuticallyacceptable salt. In another embodiment, a sub-physiologicalconcentration of salt is less than about 70 mM, less than about 60 mM,less than about 50 mM, less than about 40 mM, less than about 30 mM,less than about 20 mM, or less than about 10 mM of a pharmaceuticallyacceptable salt.

As used herein, the terms “treat” or “treatment” of a state, disorder orcondition include: (1) preventing, delaying, or reducing the incidenceand/or likelihood of the appearance of at least one clinical orsub-clinical symptom of the state, disorder or condition developing in asubject that may be afflicted with or predisposed to the state, disorderor condition, but does not yet experience or display clinical orsubclinical symptoms of the state, disorder or condition; or (2)inhibiting the state, disorder or condition, i.e., arresting, reducingor slowing the development/progression of the disease or a relapsethereof or at least one clinical or sub-clinical symptom thereof; or (3)relieving the disease, i.e., causing regression of the state, disorderor condition or at least one of its clinical or sub-clinical symptoms.As used these terms, do not necessarily imply 100% or completetreatment. Rather, there are varying degrees of treatment of which oneof ordinary skill in the art recognizes as having a potential benefit ortherapeutic effect. The benefit to a subject to be treated is eitherstatistically significant or at least perceptible to the patient or tothe physician. In this respect, the methods of treating a disorder,e.g., a bleeding disorder, of the present disclosure can provide anyamount or any level of treatment. Furthermore, the treatment provided bythe method of the present disclosure may include treatment of one ormore conditions or symptoms or signs of the disorder, being treated.

As used herein, a “therapeutically effective amount or dose” or“sufficient amount or dose” refers to a dose that produces effects forwhich it is administered. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,Gennaro, Ed., Lippincott, Williams & Wilkins).

The terms “patient” and “subject” are used interchangeably and are usedin their conventional sense to refer to a living organism suffering fromor prone to a condition that can be prevented or treated byadministration of a composition of the present disclosure, and includesboth humans and non-human animals. Examples of subjects include, but arenot limited to, humans, chimpanzees and other apes and monkey species;farm animals such as cattle, sheep, pigs, goats and horses; domesticmammals such as dogs and cats; laboratory animals including rodents suchas mice, rats and guinea pigs; birds, including domestic, wild and gamebirds such as chickens, turkeys and other gallinaceous birds, ducks,geese, and the like. The term does not denote a particular age. Thus,adult, juvenile and newborn individuals are of interest.

As used herein, “storage” means that a formulation is not immediatelyadministered to a subject once prepared, but is kept for a period oftime under particular conditions (e.g. particular temperature, etc.)prior to use. For example, a liquid or lyophilized formulation can bekept for days, weeks, months or years, prior to administration to asubject under varied temperatures such as refrigerated (0° to 10° C.) orroom temperature (e.g., temperature up to 32° C.).

As used herein, the term “about” denotes an approximate range of plus orminus 10% from a specified value. For instance, the language “about 20%”encompasses a range of 18-22%. As used herein, about also includes theexact amount. Hence “about 20%” means “about 20%” and also “20%.

As used herein, the term “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

As used herein, the term “or” refers to any one member of a particularlist and also includes any combination of members of that list.

As used herein, the terms “a” and “an” and “the” and similar referentsin the context of describing the disclosure (especially in the contextof the following claims) are to be construed to cover both the singularand the plural, unless otherwise indicated herein or clearlycontradicted by context. The terms “comprising,” “having,” “including,”and “containing” are to be construed as open-ended terms (i.e., meaning“including, but not limited to,”) unless otherwise noted.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range and each endpoint, unless otherwise indicatedherein, and each separate value and endpoint is incorporated into thespecification as if it were individually recited herein.

AAV Compositions and Formulations

In exemplary aspects, the pharmaceutical composition of the presentdisclosure comprises: adeno-associated virus (AAV) and about 5 mM toabout 25 mM of a buffering agent, about 0 mM to about 150 mM of apharmaceutically acceptable salt, about 0.001% (w/v) to about 0.01%(w/v) of a non-ionic surfactant, and about 1% (w/v) to about 10% (w/v)of a sugar or sugar alcohol.

In exemplary aspects, the pharmaceutical composition of the presentdisclosure comprises: adeno-associated virus (AAV) and about 5 mM toabout 25 mM of a buffering agent, about 50 mM to about 150 mM of apharmaceutically acceptable salt, about 0.001% (w/v) to about 0.01%(w/v) of a non-ionic surfactant, and about 1% (w/v) to about 10% (w/v)of a sugar or sugar alcohol.

In exemplary aspects, the pharmaceutical composition of the presentdisclosure comprises: adeno-associated virus (AAV) and about 5 mM toabout 25 mM of a buffering agent, about 0 mM to less than 100 mM of apharmaceutically acceptable salt, about 0.001% (w/v) to about 0.01%(w/v) of a non-ionic surfactant, and about 1% (w/v) to about 10% (w/v)of a sugar or sugar alcohol.

In exemplary aspects, the pharmaceutical composition of the presentdisclosure comprises: adeno-associated virus (AAV) and about 5 mM toabout 25 mM of a buffering agent, about 30 mM to less than 100 mM of apharmaceutically acceptable salt, about 0.001% (w/v) to about 0.01%(w/v) of a non-ionic surfactant, and about 1% (w/v) to about 10% (w/v)of a sugar or sugar alcohol.

In certain instances, the composition is a sterile composition. By“sterile” is meant that there are substantially no immunogeniccomponents in the composition, such as for example substantially nomicrobes (e.g., fungi, bacteria, viruses, spore forms, etc.).

In some embodiments, the invention provides a liquid formulation. Incertain embodiments, the formulation is lyophilized from a liquidformulation.

In exemplary aspects, the pharmaceutical composition of the presentdisclosure comprises about 5 mM to about 25 mM, about 5 mM to about 15mM, about 10 mM to about 20 mM, or about 15 mM to about 25 mM of abuffering agent. In exemplary aspects, the pharmaceutical compositioncomprises about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM,about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM,about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM of abuffering agent.

Pharmaceutically acceptable buffering agents are well known in the art,and include without limitation, phosphate buffers, histidine, sodiumcitrate, HEPES, Tris, Bicine, glycine, N-glycylglycine, sodium acetate,sodium carbonate, glycyl glycine, lysine, arginine, sodium phosphate,and mixtures thereof. In certain embodiments, the buffer is histidine(e.g., L-histidine).

In exemplary aspects, the pharmaceutical composition of the presentdisclosure comprises about 5 mM to about 25 mM, about 5 mM to about 15mM, about 10 mM to about 20 mM, or about 15 mM to about 25 mML-histidine. In exemplary aspects, the pharmaceutical compositioncomprises about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM,about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM,about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mML-histidine. In certain embodiments, the pharmaceutical compositioncomprises about 10 mM L-histidine. In certain embodiments, thepharmaceutical composition comprises about 20 mM L-histidine.

In exemplary aspects, the pharmaceutical composition of the presentdisclosure comprises about 0 mM to about 150 mM, about 5 mM to about 150mM, about 5 mM to about 100 mM, about 5 mM to about 90 mM, about 5 mM toabout 80 mM, about 5 mM to about 70 mM, about 5 mM to about 60 mM, about5 mM to about 50 mM, about 5 mM to about 40 mM, 5 mM to about 30 mM,about 30 mM to about 100 mM, about 30 mM to about 90 mM, about 30 mM toabout 80 mM, about 30 mM to about 70 mM, about 30 mM to about 60 mM,about 30 mM to about 50 mM, about 30 mM to about 40 mM, about 50 mM toabout 150 mM, 50 mM to about 120 mM, about 55 mM to about 100 mM, about60 mM to about 100 mM, about 60 mM to about 80 mM, about 70 mM to about100 mM, or about 70 mM to about 80 mM of a pharmaceutically acceptablesalt (as defined above). In certain embodiments, the pharmaceuticalcomposition of the present disclosure comprises about 80 mM to about 120mM, about 70 mM to about 90 mM, about 50 mM to about 70 mM, or about 20mM to about 40 mM of a pharmaceutically acceptable salt. In exemplaryaspects, the pharmaceutical composition of the present disclosurecomprises about 0 mM to about 100 mM, about 0 mM to about 80 mM, about 0mM to about 70 mM, about 0 mM to about 60 mM, about 0 mM to about 50 mM,about 0 mM to about 40 mM, about 0 mM to about 30 mM, about 0 mM toabout 20 mM, about 0 mM to about 10 mM, about 0 mM to about 5 mM of apharmaceutically acceptable salt. In exemplary aspects, thepharmaceutical composition comprises about 0 mM, about 5 mM, about 10mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM,about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM,about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM,about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM,about 145 mM, or about 150 mM of a pharmaceutically acceptable salt. Inother embodiments, the pharmaceutical composition comprises about 0 mMto less than about 100 mM, about 5 mM to less than about 100 mM, about10 mM to less than about 100 mM, about 20 mM to less than about 100 mM,about 30 mM to less than about 100 mM, about 40 mM to less than about100 mM, about 50 mM to less than about 100 mM, about 60 mM to less thanabout 100 mM, or about 70 mM to less than about 100 mM of apharmaceutically acceptable salt. In certain embodiments, thepharmaceutical composition of the present disclosure comprises about 0mM of a pharmaceutically acceptable salt. In certain embodiments, thepharmaceutical composition of the present disclosure comprises about 30mM of a pharmaceutically acceptable salt. In certain embodiments, thepharmaceutical composition of the present disclosure comprises about 40mM of a pharmaceutically acceptable salt. In certain embodiments, thepharmaceutical composition of the present disclosure comprises about 50mM of a pharmaceutically acceptable salt. In certain embodiments, thepharmaceutical composition of the present disclosure comprises about 60mM of a pharmaceutically acceptable salt. In certain embodiments, thepharmaceutical composition of the present disclosure comprises about 70mM of a pharmaceutically acceptable salt. In certain embodiments, thepharmaceutical composition of the present disclosure comprises about 80mM of a pharmaceutically acceptable salt. In certain embodiments, thepharmaceutical composition of the present disclosure comprises about 100mM of a pharmaceutically acceptable salt. In certain embodiments, thepharmaceutically acceptable salt is a sodium salt (e.g., sodiumchloride).

Advantageously, it has been found that pharmaceutical compositionscontaining a sub-physiological concentration of a pharmaceuticallyacceptable salt forms compact lyocakes with lower moisture content andassists in the formation of an improved lyocake. In certain embodiments,the sub-physiological concentration of the pharmaceutically acceptablesalt results in white, compact and homogenous lyocakes. Accordingly, incertain embodiments, the present disclosure provides low saltpharmaceutical compositions containing a sub-physiological concentrationof a pharmaceutically acceptable salt, for example, less than about 100mM of a pharmaceutically acceptable salt. In one embodiment, a low saltformulation provided herein contains less than about 100 mM of apharmaceutical salt. In certain embodiments, a low salt pharmaceuticalcomposition provided herein contains less than about 80 mM of apharmaceutical salt. In certain embodiments, a low salt pharmaceuticalcomposition provided herein contains less than about 70 mM of apharmaceutical salt. In another embodiment, a low salt pharmaceuticalcomposition provided herein contains less than about 60 mM of apharmaceutical salt. In another embodiment, a low salt pharmaceuticalcomposition provided herein contains less than about 50 mM of apharmaceutical salt. In another embodiment, a low salt pharmaceuticalcomposition provided herein contains less than about 40 mM of apharmaceutical salt. In another embodiment, a low salt pharmaceuticalcomposition provided herein contains less than about 30 mM of apharmaceutical salt. In another embodiment, a low salt pharmaceuticalcomposition provided herein contains less than about 20 mM of apharmaceutical salt. In another embodiment, a low salt pharmaceuticalcomposition provided herein contains less than about 10 mM of apharmaceutical salt. In another embodiment, a low salt pharmaceuticalcomposition contains between about 30 mM and about 60 mM of apharmaceutically acceptable salt. In yet other embodiments, a low saltpharmaceutical composition contains about 0 mM, about 5 mM, about 10 mM,about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM,about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about95 mM, or about 100 mM of a pharmaceutically acceptable salt. In certainembodiments, the pharmaceutical composition of the present disclosurecomprises about 0 mM of a pharmaceutically acceptable salt. In certainembodiments, the pharmaceutical composition of the present disclosurecomprises about 30 mM of a pharmaceutically acceptable salt. In certainembodiments, the pharmaceutical composition of the present disclosurecomprises about 40 mM of a pharmaceutically acceptable salt. In certainembodiments, the pharmaceutical composition of the present disclosurecomprises about 50 mM of a pharmaceutically acceptable salt. In certainembodiments, the pharmaceutical composition of the present disclosurecomprises about 60 mM of a pharmaceutically acceptable salt. In certainembodiments, the pharmaceutical composition of the present disclosurecomprises about 70 mM of a pharmaceutically acceptable salt. In certainembodiments, the pharmaceutical composition of the present disclosurecomprises about 80 mM of a pharmaceutically acceptable salt. In apreferred embodiment, a low salt pharmaceutical composition is alyophilized formulation. In a preferred embodiment, the salt is a sodiumsalt (e.g., sodium chloride).

In exemplary aspects, the pharmaceutical composition of the presentdisclosure comprises about 0 mM to about 150 mM, about 5 mM to about 150mM, about 5 mM to about 100 mM, about 5 mM to about 90 mM, about 5 mM toabout 80 mM, about 5 mM to about 70 mM, about 5 mM to about 60 mM, about5 mM to about 50 mM, about 5 mM to about 40 mM, 5 mM to about 30 mM,about 30 mM to about 100 mM, about 30 mM to about 90 mM, about 30 mM toabout 80 mM, about 30 mM to about 70 mM, about 30 mM to about 60 mM,about 30 mM to about 50 mM, about 30 mM to about 40 mM, about 50 mM toabout 150 mM, 50 mM to about 120 mM, about 55 mM to about 100 mM, about60 mM to about 100 mM, about 60 mM to about 80 mM, about 70 mM to about100 mM, or about 70 mM to about 80 mM sodium chloride. In certainembodiments, the pharmaceutical composition of the present disclosurecomprises about 80 mM to about 120 mM, about 70 mM to about 90 mM, about50 mM to about 70 mM, or about 20 mM to about 40 mM sodium chloride. Inexemplary aspects, the pharmaceutical composition of the presentdisclosure comprises about 0 mM to about 100 mM, about 0 mM to about 80mM, about 0 mM to about 70 mM, about 0 mM to about 60 mM, about 0 mM toabout 50 mM, about 0 mM to about 40 mM, about 0 mM to about 30 mM, about0 mM to about 20 mM, about 0 mM to about 10 mM, about 0 mM to about 5 mMsodium chloride. In certain embodiments, the pharmaceutical compositioncomprises less than about 100 mM, less than about 80 mM, less than about70 mM, or less than about 60 mM sodium chloride. In other embodiments,the pharmaceutical composition comprises about 0 mM to less than about100 mM, about 5 mM to less than about 100 mM, about 10 mM to less thanabout 100 mM, about 20 mM to less than about 100 mM, about 30 mM to lessthan about 100 mM, about 40 mM to less than about 100 mM, about 50 mM toless than about 100 mM, about 60 mM to less than about 100 mM, or about70 mM to less than about 100 mM sodium chloride. In exemplary aspects,the pharmaceutical composition comprises about 0 mM, about 5 mM, about10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM,about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM,about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM,about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM,about 145 mM, or about 150 mM sodium chloride. In certain embodiments,the pharmaceutical composition of the present disclosure comprises about0 mM sodium chloride. In certain embodiments, the pharmaceuticalcomposition of the present disclosure comprises about 10 mM sodiumchloride. In certain embodiments, the pharmaceutical composition of thepresent disclosure comprises about 20 mM sodium chloride. In certainembodiments, the pharmaceutical composition of the present disclosurecomprises about 30 mM sodium chloride. In certain embodiments, thepharmaceutical composition of the present disclosure comprises about 40mM sodium chloride. In certain embodiments, the pharmaceuticalcomposition of the present disclosure comprises about 50 mM sodiumchloride. In certain embodiments, the pharmaceutical composition of thepresent disclosure comprises about 60 mM sodium chloride. In certainembodiments, the pharmaceutical composition of the present disclosurecomprises about 70 mM sodium chloride. In certain embodiments, thepharmaceutical composition of the present disclosure comprises about 80mM sodium chloride. In certain embodiments, the pharmaceuticalcomposition of the present disclosure comprises about 100 mM sodiumchloride.

Advantageously, it was also found that the inclusion of a non-ionicsurfactant substantially reduces the loss of AAV on surfaces andprevents formation of visible particles of the pharmaceuticalcompositions. Accordingly, in certain embodiments, pharmaceuticalcompositions containing a stabilizing concentration of a non-ionicdetergent are provided. Pharmaceutically acceptable non-ionicsurfactants that may be used in the formulations disclosed herein areknown in the art of pharmaceutical science, and include, withoutlimitation, Polysorbate 80 (Tween 80; PS80), Polysorbate 20 (Tween 20;PS20), and various poloxamers or pluronics, including Pluronic F-68, andBRIJ 35, or mixtures thereof. In a preferred embodiment, the non-ionicsurfactant used in the present pharmaceutical compositions isPolysorbate 80. In exemplary aspects, the pharmaceutical composition ofthe present disclosure comprises Super Refined™ PS80 commerciallyavailable from Croda Health Care (Snaith, UK).

In certain embodiments, the pharmaceutical composition of the presentdisclosure comprises about 0.001% (w/v) to about 0.01% (w/v) or about0.0025% (w/v) to about 0.0075% (w/v) non-ionic surfactant. In exemplaryaspects, the pharmaceutical composition comprises about 0.001% (w/v),about 0.0015% (w/v), about 0.002% (w/v), about 0.0025% (w/v), about0.003% (w/v), about 0.0035% (w/v), about 0.004% (w/v), about 0.0045%(w/v), about 0.005% (w/v), about 0.0055% (w/v), about 0.006% (w/v),about 0.0065% (w/v), about 0.007% (w/v), about 0.0075% (w/v), about0.008% (w/v), about 0.0085% (w/v), about 0.009% (w/v), about 0.0095%(w/v), about 0.001% (w/v) non-ionic surfactant. In certain embodiments,the pharmaceutical composition of the present disclosure comprises about0.005% (w/v) non-ionic surfactant.

In exemplary aspects, the pharmaceutical composition of the presentdisclosure comprises about 0.001% (w/v) to about 0.01% (w/v) or about0.0025% (w/v) to about 0.0075% (w/v) polysorbate 80 (e.g., SuperRefined™ PS80). In exemplary aspects, the pharmaceutical compositioncomprises about 0.001% (w/v), about 0.0015% (w/v), about 0.002% (w/v),about 0.0025% (w/v), about 0.003% (w/v), about 0.0035% (w/v), about0.004% (w/v), about 0.0045% (w/v), about 0.005% (w/v), about 0.0055%(w/v), about 0.006% (w/v), about 0.0065% (w/v), about 0.007% (w/v),about 0.0075% (w/v), about 0.008% (w/v), about 0.0085% (w/v), about0.009% (w/v), about 0.0095% (w/v), about 0.001% (w/v) PS80. In certainembodiments, the pharmaceutical composition of the present disclosurecomprises about 0.005% (w/v) PS80.

It was found that the inclusion of moderate levels (i.e., between about1% to about 10%) of one or more sugar and/or sugar alcohol assists inthe stability of the liquid and/or lyophilized formulations. Forexample, the sugar and/or sugar alcohol allows for better propertiesduring freeze/thawing cycles. Accordingly, in certain embodiments, thepresent invention provides pharmaceutical compositions containingbetween about 2% and about 10% of one or more sugars and/or sugaralcohols. Any sugar such as mono-, di-, or polysaccharides, orwater-soluble glucans, including for example fructose, glucose, mannose,sorbose, xylose, maltose, lactose, sucrose, dextran, trehalose,pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch,and carboxymethylcellulose may be used. In a particular embodiment, thesugar is sucrose, trehalose, or a combination thereof. In certainembodiments, the trehalose is trehalose dihydrate. Sugar alcohols aredefined as a hydrocarbon having between about 4 and about 8 carbon atomsand a hydroxyl group. Non-limiting examples of sugar alcohols that maybe used in the pharmaceutical compositions provided herein include,mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, andarabitol. In certain embodiments, mannitol is used as a sugar alcoholadditive. In certain embodiments, a pharmaceutical composition containsboth a sugar and a sugar alcohol additive.

The sugars and sugar alcohols may be used individually or incombination. In some embodiments, the sugar, sugar alcohol, orcombination thereof will be present in the formulation at aconcentration of about 1% to about 10% (w/v), about 1% (w/v) to about1.5% (w/v), about 2.5% to about 7.5% (w/v), or about 1% to about 5%(w/v). In exemplary aspects, the pharmaceutical composition of thepresent disclosure comprises about 1.0% (w/v), about 1.1% (w/v), about1.2% (w/v), about 1.3% (w/v), about 1.4% (w/v), about 1.5% (w/v), about1.6% (w/v), about 1.7% (w/v), about 1.8% (w/v), about 1.9% (w/v), about2.0% (w/v), about 2.5% (w/v), about 3.0% (w/v), about 3.5% (w/v), about4.0% (w/v), about 4.5% (w/v), about 5.0% (w/v), about 5.5% (w/v), about6.0% (w/v), about 6.5% (w/v), about 7.0% (w/v), about 7.5% (w/v), about8.0% (w/v), about 8.5% (w/v), about 9.0% (w/v), about 9.5% (w/v), orabout 10% (w/v) sugar, sugar alcohol, or combination thereof. In certainembodiments, the sugar is sucrose, trehalose, or a combination thereof.In certain embodiments, the trehalose is trehalose dihydrate.

In certain embodiments, the pharmaceutical composition of the presentdisclosure comprises about 1% to about 10% (w/v), about 1% (w/v) toabout 1.5% (w/v), about 2.5% to about 7.5% (w/v), or about 1% to about5% (w/v) sucrose, trehalose, or a combination thereof. In exemplaryaspects, the pharmaceutical composition of the present disclosurecomprises about 1.0% (w/v), about 1.1% (w/v), about 1.2% (w/v), about1.3% (w/v), about 1.4% (w/v), about 1.5% (w/v), about 1.6% (w/v), about1.7% (w/v), about 1.8% (w/v), about 1.9% (w/v), about 2.0% (w/v), about2.5% (w/v), about 3.0% (w/v), about 3.5% (w/v), about 4.0% (w/v), about4.5% (w/v), about 5.0% (w/v), about 5.5% (w/v), about 6.0% (w/v), about6.5% (w/v), about 7.0% (w/v), about 7.5% (w/v), about 8.0% (w/v), about8.5% (w/v), about 9.0% (w/v), about 9.5% (w/v), or about 10% (w/v)sucrose, trehalose, or a combination thereof. In certain embodiments,the pharmaceutical composition of the present disclosure comprises about1.3% (w/v) sucrose, trehalose, or a combination thereof. In certainembodiments, the pharmaceutical composition of the present disclosurecomprises about 5% (w/v) sucrose, trehalose, or a combination thereof.In exemplary aspects, the pharmaceutical composition of the presentdisclosure comprises 10 mM to about 150 mM, about 30 mM to about 40 mM,or about 35 mM to about 132 mM sucrose, trehalose, or a combinationthereof. In exemplary aspects, the pharmaceutical composition of thepresent disclosure comprises about 10 mM, about 15 mM, about 20 mM,about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM,about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about110 mM, about 120 mM, about 130 mM, about 140 mM, or about 150 mMsucrose, trehalose, or a combination thereof. In certain embodiments,the pharmaceutical composition of the present disclosure comprises about35 mM sucrose, trehalose, or a combination thereof. In certainembodiments, the pharmaceutical composition of the present disclosurecomprises about 132 mM sucrose, trehalose, or a combination thereof. Inexemplary aspects, the trehalose is trehalose dihydrate.

In exemplary aspects, the pharmaceutical composition of the presentdisclosure comprises: adeno-associated virus (AAV) and about 5 mM toabout 25 mM L-histidine, about 0 mM to about 150 mM pharmaceuticallyacceptable salt, about 0.001% (w/v) to about 0.01% (w/v) non-ionicsurfactant, and about 1% (w/v) to about 10% (w/v) sugar and/or sugaralcohol. In certain embodiments, the pharmaceutically acceptable salt isabout 0 nM to about 100 mM. In certain embodiments, the pharmaceuticallyacceptable salt is about 30 nM to about 100 mM. In certain embodiments,the pharmaceutically acceptable salt is about 5 nM to about 150 mM. Incertain embodiments, the pharmaceutically acceptable salt is about 50 nMto about 150 mM. In certain embodiments, the pharmaceutically acceptablesalt is sodium chloride.

In exemplary aspects, the pharmaceutical composition of the presentdisclosure further comprises glycine. In exemplary aspects, thepharmaceutical composition of the present disclosure comprises about 30mM to about 70 mM, about 35 mM to about 65 mM glycine, about 40 mM toabout 60 mM, or about 45 mM to about 55 mM glycine. In exemplaryaspects, the pharmaceutical composition of the present disclosurecomprises about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50mM, about 55 mM, about 60 mM, about 65 mM, or about 70 mM glycine. Incertain embodiments, the pharmaceutical composition of the presentdisclosure further comprises about 50 mM glycine.

In exemplary aspects, the pharmaceutical composition of the presentdisclosure further comprises mannitol. In exemplary aspects, thepharmaceutical composition of the present disclosure comprises about 50mM to about 150 mM, about 60 mM to about 140 mM, about 70 mM to about130 mM, about 80 mM to about 120 mM, or about 90 mM to about 110 mMmannitol. In certain embodiments, the pharmaceutical composition of thepresent disclosure comprises about 110 mM mannitol. In exemplaryaspects, the pharmaceutical composition of the present disclosurecomprises glycine or mannitol, but not both glycine and mannitol.

In other embodiments, the pharmaceutical composition of the presentdisclosure comprises: adeno-associated virus (AAV) and about 5 mM toabout 25 mM L-histidine, about 0 mM to about 150 mM pharmaceuticallyacceptable salt, about 0.001% (w/v) to about 0.01% (w/v) non-ionicsurfactant, and about 1% (w/v) to about 10% (w/v) sugar and/or sugaralcohol. In certain embodiments, the pharmaceutically acceptable salt isabout 0 nM to about 100 mM. In certain embodiments, the pharmaceuticallyacceptable salt is about 30 nM to about 100 mM. In certain embodiments,the pharmaceutically acceptable salt is about 5 nM to about 150 mM. Incertain embodiments, the pharmaceutically acceptable salt is about 50 nMto about 150 mM. certain embodiments, the sugar and/or sugar alcohol issucrose, trehalose, mannitol, and a combination thereof. In certainembodiments, the pharmaceutically acceptable salt is sodium chloride.

In other embodiments, the pharmaceutical composition of the presentdisclosure comprises: adeno-associated virus (AAV) and about 5 mM toabout 25 mM L-histidine, about 0 mM to about 150 mM pharmaceuticallyacceptable salt, about 0.001% (w/v) to about 0.01% (w/v) non-ionicsurfactant, and about 1% (w/v) to about 10% (w/v) sugar and/or sugaralcohol. In certain embodiments, the pharmaceutically acceptable salt isabout 0 nM to about 100 mM. In certain embodiments, the pharmaceuticallyacceptable salt is about 30 nM to about 100 mM. In certain embodiments,the pharmaceutically acceptable salt is about 5 nM to about 150 mM. Incertain embodiments, the pharmaceutically acceptable salt is about 50 nMto about 150 mM. In certain embodiments, the non-ionic surfactant isPolysorbate 20, Polysorbate 80, Pluronic F-68, BRIJ 35, and acombination thereof. In certain embodiments, the sugar and/or sugaralcohol is sucrose, trehalose, mannitol, and a combination thereof. Incertain embodiments, the non-ionic surfactant is PS80. In certainembodiments, the pharmaceutically acceptable salt is sodium chloride.

In exemplary aspects, the pharmaceutical composition of the presentdisclosure comprises: adeno-associated virus (AAV) and about 5 mM toabout 25 mM L-histidine, less than about 100 mM pharmaceuticallyacceptable salt, about 0.001% (w/v) to about 0.01% (w/v) non-ionicsurfactant, and about 1% (w/v) to about 10% (w/v) sugar and/or sugaralcohol. In certain embodiments, the pharmaceutically acceptable salt isabout 0 nM to less than about 100 mM. In certain embodiments, thepharmaceutically acceptable salt is about 0 nM to about 80 mM, about 0nM to about 70 mM, about 0 mM to about 60 mM, about 0 mM to about 50 mM,about 0 mM to about 40 mM, 0 mM to about 30 mM, about 0 mM to about 20mM, or 0 mM to about 10 mM. In certain embodiments, the pharmaceuticallyacceptable salt is about 30 nM to less than about 100 mM. In certainembodiments, the pharmaceutically acceptable salt is about 30 nM toabout 80 mM, about 30 nM to about 70 mM, about 30 mM to about 60 mM,about 30 mM to about 50 mM, or about 30 mM to about 40 mM. In certainembodiments, the pharmaceutically acceptable salt is sodium chloride. Incertain embodiments, the pharmaceutical composition of the presentdisclosure comprises glycine or mannitol, but not both glycine and/ormannitol. In certain embodiments, the pharmaceutical composition of thepresent disclosure comprises glycine or mannitol, but not both glycineand mannitol. In certain embodiments, the formulation is a liquidformulation. In certain embodiments, the formulation is a lyophilizedformulation. In certain embodiments, the lower salt pharmaceuticalcomposition is a lyophilized formulation.

In other embodiments, the pharmaceutical composition of the presentdisclosure comprises: adeno-associated virus (AAV) and about 5 mM toabout 25 mM L-histidine, less than about 100 mM pharmaceuticallyacceptable salt, about 0.001% (w/v) to about 0.01% (w/v) non-ionicsurfactant, and about 1% (w/v) to about 10% (w/v) sugar and/or sugaralcohol. In certain embodiments, the pharmaceutically acceptable salt isabout 0 nM to less than about 100 mM. In certain embodiments, thepharmaceutically acceptable salt is about 0 nM to about 80 mM, about 0nM to about 70 mM, about 0 mM to about 60 mM, about 0 mM to about 50 mM,about 0 mM to about 40 mM, 0 mM to about 30 mM, about 0 mM to about 20mM, or 0 mM to about 10 mM. In certain embodiments, the pharmaceuticallyacceptable salt is about 30 nM to less than about 100 mM. In certainembodiments, the pharmaceutically acceptable salt is about 30 nM toabout 80 mM, about 30 nM to about 70 mM, about 30 mM to about 60 mM,about 30 mM to about 50 mM, or about 30 mM to about 40 mM. In certainembodiments, the sugar and/or sugar alcohol is sucrose, trehalose,mannitol, and a combination thereof. In certain embodiments, thepharmaceutically acceptable salt is sodium chloride. In certainembodiments, the formulation is a liquid formulation. In certainembodiments, the formulation is a lyophilized formulation. In certainembodiments, the lower salt pharmaceutical composition is a lyophilizedformulation.

In other embodiments, the pharmaceutical composition of the presentdisclosure comprises: adeno-associated virus (AAV) and about 5 mM toabout 25 mM L-histidine, less than about 100 mM pharmaceuticallyacceptable salt, about 0.001% (w/v) to about 0.01% (w/v) non-ionicsurfactant, and about 1% (w/v) to about 10% (w/v) sugar and/or sugaralcohol. In certain embodiments, the pharmaceutically acceptable salt isabout 0 nM to less than about 100 mM. In certain embodiments, thepharmaceutically acceptable salt is about 0 nM to about 80 mM, about 0nM to about 70 mM, about 0 mM to about 60 mM, about 0 mM to about 50 mM,about 0 mM to about 40 mM, 0 mM to about 30 mM, about 0 mM to about 20mM, or 0 mM to about 10 mM. In certain embodiments, the pharmaceuticallyacceptable salt is about 30 nM to less than about 100 mM. In certainembodiments, the pharmaceutically acceptable salt is about 30 nM toabout 80 mM, about 30 nM to about 70 mM, about 30 mM to about 60 mM,about 30 mM to about 50 mM, or about 30 mM to about 40 mM. In certainembodiments, the non-ionic surfactant is Polysorbate 20, Polysorbate 80,Pluronic F-68, BRIJ 35, and a combination thereof. In certainembodiments, the sugar and/or sugar alcohol is sucrose, trehalose,mannitol, and a combination thereof. In certain embodiments, thenon-ionic surfactant is PS80. In certain embodiments, thepharmaceutically acceptable salt is sodium chloride. In certainembodiments, the formulation is a liquid formulation. In certainembodiments, the formulation is a lyophilized formulation. In certainembodiments, the lower salt pharmaceutical composition is a lyophilizedformulation.

In exemplary aspects, the pharmaceutical composition of the presentdisclosure comprises: adeno-associated virus (AAV) and about 5 mM toabout 25 mM L-histidine, about 50 mM to about 150 mM sodium chloride,about 0.001% (w/v) to about 0.01% (w/v) polysorbate 80 (PS80), and about1% (w/v) to about 10% (w/v) sucrose or trehalose.

In exemplary aspects, the pharmaceutical composition of the presentdisclosure comprises: adeno-associated virus (AAV) and about 5 mM toabout 25 mM L-histidine, about 0 mM to less than about 100 mM sodiumchloride, about 0.001% (w/v) to about 0.01% (w/v) polysorbate 80 (PS80),and about 1% (w/v) to about 10% (w/v) sucrose or trehalose. In certainembodiments, the formulation is a liquid formulation. In certainembodiments, the formulation is a lyophilized formulation. In certainembodiments, the lower salt pharmaceutical composition is a lyophilizedformulation.

In exemplary aspects, the pharmaceutical composition of the presentdisclosure comprises: adeno-associated virus (AAV) and about 5 mM toabout 25 mM L-histidine, about 30 mM to about 80 mM sodium chloride,about 0.001% (w/v) to about 0.01% (w/v) polysorbate 80 (PS80), and about1% (w/v) to about 10% (w/v) sucrose or trehalose. In certainembodiments, the formulation is a liquid formulation. In certainembodiments, the formulation is a lyophilized formulation. In certainembodiments, the lower salt pharmaceutical composition is a lyophilizedformulation.

In certain embodiments, lower salt pharmaceutical compositions areprovided comprising adeno-associated virus (AAV) and about 5 mM to about25 mM L-histidine, about 50 mM to about 150 mM sodium chloride, about0.001% (w/v) to about 0.01% (w/v) polysorbate 80 (PS80), and about 1%(w/v) to about 10% (w/v) sucrose or trehalose. In certain embodiments,the pharmaceutical composition of the present disclosure furthercomprises about 30 mM to about 70 mM glycine. In certain embodiments,the formulation is a liquid formulation. In certain embodiments, theformulation is a lyophilized formulation. In certain embodiments, thelower salt pharmaceutical composition is a lyophilized formulation.

In certain embodiments, lower salt pharmaceutical compositions areprovided comprising adeno-associated virus (AAV) and about 5 mM to about25 mM L-histidine, about 0 mM to less than about 100 mM sodium chloride,about 0.001% (w/v) to about 0.01% (w/v) polysorbate 80 (PS80), and about1% (w/v) to about 10% (w/v) sucrose or trehalose. In certainembodiments, the pharmaceutical composition of the present disclosurefurther comprises about 30 mM to about 70 mM glycine. In certainembodiments, the pharmaceutical composition comprises less than about 80mM sodium chloride. In certain embodiments, the pharmaceuticalcomposition comprises less than about 70 mM sodium chloride. In certainembodiments, the pharmaceutical composition comprises less than about 60mM sodium chloride. In certain embodiments, the pharmaceuticalcomposition comprises less than about 50 mM sodium chloride. In certainembodiments, the pharmaceutical composition comprises less than about 40mM sodium chloride. In certain embodiments, the pharmaceuticalcomposition comprises less than about 30 mM sodium chloride. In certainembodiments, the pharmaceutical composition comprises about 0 mM toabout 80 mM sodium chloride. In certain embodiments, the pharmaceuticalcomposition comprises about 0 mM to about 70 mM sodium chloride. Incertain embodiments, the pharmaceutical composition comprises about 0 mMto about 60 mM sodium chloride. In certain embodiments, thepharmaceutical composition comprises about 0 mM to about 50 mM sodiumchloride. In certain embodiments, the pharmaceutical compositioncomprises about 0 mM to about 40 mM sodium chloride. In certainembodiments, the pharmaceutical composition comprises about 0 mM toabout 30 mM sodium chloride. In certain embodiments, the pharmaceuticalcomposition comprises about 30 mM to about 80 mM sodium chloride. Incertain embodiments, the pharmaceutical composition comprises about 30mM to about 70 mM sodium chloride. In certain embodiments, thepharmaceutical composition comprises about 30 mM to about 60 mM sodiumchloride. In certain embodiments, the pharmaceutical compositioncomprises about 30 mM to about 50 mM sodium chloride. In certainembodiments, the pharmaceutical composition comprises about 30 mM toabout 40 mM sodium chloride. In certain embodiments, the formulation isa liquid formulation. In certain embodiments, the formulation is alyophilized formulation. In certain embodiments, the lower saltpharmaceutical composition is a lyophilized formulation.

The present disclosure also provides a pharmaceutical compositioncomprising adeno-associated virus (AAV) and about 20 mM L-histidine,about 70 mM sodium chloride, about 0.005% (w/v) polysorbate 80 (PS80),and about 5% (w/v) sucrose. In certain embodiments, the pH of thepharmaceutical composition is about 7.5±0.2 and/or the osmolality isabout 300±10 mOsmol/kg.

The present disclosure further provides a pharmaceutical compositioncomprising adeno-associated virus (AAV) and about 20 mM L-histidine,about 60 mM sodium chloride, about 0.005% (w/v) polysorbate 80 (PS80),about 35 mM trehalose, and about 110 mM mannitol. In certainembodiments, the pH of the pharmaceutical composition is about 7.0±0.2and/or the osmolality is about 390±10 mOsmol/kg. In certain embodiments,the pH of the pharmaceutical composition is about 7.0±0.2 and/or theosmolality is about 290±10 mOsmol/kg.

The present disclosure further provides a pharmaceutical compositioncomprising adeno-associated virus (AAV) and about 10 mM L-histidine,about 100 mM sodium chloride, about 0.005% (w/v) polysorbate 80 (PS80),about 5% (w/v) trehalose, and about 50 mM glycine. In certainembodiments, the pH of the pharmaceutical composition is about 7.0±0.2and/or the osmolality is about 400±10 mOsmol/kg.

The present disclosure further provides a pharmaceutical compositionthat can be liquid or lyophilized (e.g., lyophilized from a liquidformulation) comprising adeno-associated virus (AAV) and about 10 mML-histidine, about 80 mM sodium chloride, about 0.005% (w/v) polysorbate80 (PS80), about 5% (w/v) trehalose, and about 50 mM glycine. In certainembodiments, the pH of the pharmaceutical composition is about 7.0±0.2.

The present disclosure further provides a pharmaceutical compositionthat can be liquid or lyophilized (e.g., lyophilized from a liquidformulation) comprising adeno-associated virus (AAV) and about 10 mML-histidine, about 70 mM sodium chloride, about 0.005% (w/v) polysorbate80 (PS80), about 5% (w/v) trehalose, and about 50 mM glycine. In certainembodiments, the pH of the pharmaceutical composition is about 7.0±0.2.

The present disclosure further provides a pharmaceutical compositionthat can be liquid or lyophilized (e.g., lyophilized from a liquidformulation) comprising adeno-associated virus (AAV) and about 10 mML-histidine, about 60 mM sodium chloride, about 0.005% (w/v) polysorbate80 (PS80), about 5% (w/v) trehalose, and about 50 mM glycine. In certainembodiments, the pH of the pharmaceutical composition is about 7.0±0.2.

The present disclosure further provides a pharmaceutical compositionthat can be liquid or lyophilized (e.g., lyophilized from a liquidformulation) comprising adeno-associated virus (AAV) and about 10 mML-histidine, about 50 mM sodium chloride, about 0.005% (w/v) polysorbate80 (PS80), about 5% (w/v) trehalose, and about 50 mM glycine. In certainembodiments, the pH of the pharmaceutical composition is about 7.0±0.2.

The present disclosure further provides a pharmaceutical compositionthat can be liquid or lyophilized (e.g., lyophilized from a liquidformulation) comprising adeno-associated virus (AAV) and about 10 mML-histidine, about 40 mM sodium chloride, about 0.005% (w/v) polysorbate80 (PS80), about 5% (w/v) trehalose, and about 50 mM glycine. In certainembodiments, the pH of the pharmaceutical composition is about 7.0±0.2.

The present disclosure further provides a pharmaceutical compositionthat can be liquid or lyophilized (e.g., lyophilized from a liquidformulation) comprising adeno-associated virus (AAV) and about 10 mML-histidine, about 30 mM sodium chloride, about 0.005% (w/v) polysorbate80 (PS80), about 5% (w/v) trehalose, and about 50 mM glycine. In certainembodiments, the pH of the pharmaceutical composition is about 7.0±0.2.

The present disclosure further provides a pharmaceutical compositionthat can be liquid or lyophilized (e.g., lyophilized from a liquidformulation) comprising adeno-associated virus (AAV) and about 10 mML-histidine, about 20 mM sodium chloride, about 0.005% (w/v) polysorbate80 (PS80), about 5% (w/v) trehalose, and about 50 mM glycine. In certainembodiments, the pH of the pharmaceutical composition is about 7.0±0.2.

The present disclosure further provides a pharmaceutical compositionthat can be liquid or lyophilized (e.g., lyophilized from a liquidformulation) comprising adeno-associated virus (AAV) and about 10 mML-histidine, about 10 mM sodium chloride, about 0.005% (w/v) polysorbate80 (PS80), about 5% (w/v) trehalose, and about 50 mM glycine. In certainembodiments, the pH of the pharmaceutical composition is about 7.0±0.2.

The present disclosure further provides a pharmaceutical compositionthat can be liquid or lyophilized (e.g., lyophilized from a liquidformulation) comprising adeno-associated virus (AAV) and about 10 mML-histidine, about 0 mM sodium chloride, about 0.005% (w/v) polysorbate80 (PS80), about 5% (w/v) trehalose, and about 50 mM glycine. In certainembodiments, the pH of the pharmaceutical composition is about 7.0±0.2.

Additional Components

In exemplary embodiments, the formulations or pharmaceuticalcompositions of the present disclosure comprise additionalpharmaceutically acceptable ingredients. In exemplary aspects, theformulations or pharmaceutical compositions comprise any one or acombination of the following: acidifying agents, additives, adsorbents,aerosol propellants, air displacement agents, alkalizing agents,anticaking agents, anticoagulants, antimicrobial preservatives,antioxidants, antiseptics, bases, binders, buffering agents, chelatingagents, coating agents, coloring agents, desiccants, detergents,diluents, disinfectants, disintegrants, dispersing agents, dissolutionenhancing agents, dyes, emollients, emulsifying agents, emulsionstabilizers, fillers, film forming agents, flavor enhancers, flavoringagents, flow enhancers, gelling agents, granulating agents, humectants,lubricants, mucoadhesives, ointment bases, ointments, oleaginousvehicles, organic bases, pastille bases, pigments, plasticizers,polishing agents, preservatives, sequestering agents, skin penetrants,solubilizing agents, solvents, stabilizing agents, suppository bases,surface active agents, surfactants, suspending agents, sweeteningagents, therapeutic agents, thickening agents, tonicity agents, toxicityagents, viscosity-increasing agents, water-absorbing agents,water-miscible cosolvents, water softeners, or wetting agents. In someembodiments, the formulations or pharmaceutical compositions of thepresent disclosure comprise any one or a combination of the followingcomponents: acacia, acesulfame potassium, acetyltributyl citrate,acetyltriethyl citrate, agar, albumin, alcohol, dehydrated alcohol,denatured alcohol, dilute alcohol, aleuritic acid, alginic acid,aliphatic polyesters, alumina, aluminum hydroxide, aluminum stearate,amylopectin, α-amylose, ascorbic acid, ascorbyl palmitate, aspartame,bacteriostatic water for injection, bentonite, bentonite magma,benzalkonium chloride, benzethonium chloride, benzoic acid, benzylalcohol, benzyl benzoate, bronopol, butylated hydroxyanisole, butylatedhydroxytoluene, butylparaben, butylparaben sodium, calcium alginate,calcium ascorbate, calcium carbonate, calcium cyclamate, dibasicanhydrous calcium phosphate, dibasic dehydrate calcium phosphate,tribasic calcium phosphate, calcium propionate, calcium silicate,calcium sorbate, calcium stearate, calcium sulfate, calcium sulfatehemihydrate, canola oil, carbomer, carbon dioxide, carboxymethylcellulose calcium, carboxymethyl cellulose sodium, p-carotene,carrageenan, castor oil, hydrogenated castor oil, cationic emulsifyingwax, cellulose acetate, cellulose acetate phthalate, ethyl cellulose,microcrystalline cellulose, powdered cellulose, silicifiedmicrocrystalline cellulose, sodium carboxymethyl cellulose, cetostearylalcohol, cetrimide, cetyl alcohol, chlorhexidine, chlorobutanol,chlorocresol, cholesterol, chlorhexidine acetate, chlorhexidinegluconate, chlorhexidine hydrochloride, chlorodifluoroethane (HCFC),chlorodifluoromethane, chlorofluorocarbons (CFC)chlorophenoxyethanol,chloroxylenol, corn syrup solids, anhydrous citric acid, citric acidmonohydrate, cocoa butter, coloring agents, corn oil, cottonseed oil,cresol, m-cresol, o-cresol, p-cresol, croscarmellose sodium,crospovidone, cyclamic acid, cyclodextrins, dextrates, dextrin,dextrose, dextrose anhydrous, diazolidinyl urea, dibutyl phthalate,dibutyl sebacate, diethanolamine, diethyl phthalate, difluoroethane(HFC), dimethyl-β-cyclodextrin, cyclodextrin-type compounds such asCaptisol®, dimethyl ether, dimethyl phthalate, dipotassium edentate,disodium edentate, disodium hydrogen phosphate, docusate calcium,docusate potassium, docusate sodium, dodecyl gallate,dodecyltrimethylammonium bromide, edentate calcium disodium, edtic acid,eglumine, ethyl alcohol, ethylcellulose, ethyl gallate, ethyl laurate,ethyl maltol, ethyl oleate, ethylparaben, ethylparaben potassium,ethylparaben sodium, ethyl vanillin, fructose, fructose liquid, fructosemilled, fructose pyrogen-free, powdered fructose, fumaric acid, gelatin,glucose, liquid glucose, glyceride mixtures of saturated vegetable fattyacids, glycerin, glyceryl behenate, glyceryl monooleate, glycerylmonostearate, self-emulsifying glyceryl monostearate, glycerylpalmitostearate, glycine, glycols, glycofurol, guar gum,heptafluoropropane (HFC), hexadecyltrimethylammonium bromide, highfructose syrup, human serum albumin, hydrocarbons (HC), dilutehydrochloric acid, hydrogenated vegetable oil type II, hydroxyethylcellulose, 2-hydroxyethyl-β-cyclodextrin, hydroxypropyl cellulose,low-substituted hydroxypropyl cellulose, 2-hydroxypropyl-β-cyclodextrin,hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate,imidurea, indigo carmine, ion exchangers, iron oxides, isopropylalcohol, isopropyl myristate, isopropyl palmitate, isotonic saline,kaolin, lactic acid, lactitol, lactose, lanolin, lanolin alcohols,anhydrous lanolin, lecithin, magnesium aluminum silicate, magnesiumcarbonate, normal magnesium carbonate, magnesium carbonate anhydrous,magnesium carbonate hydroxide, magnesium hydroxide, magnesium laurylsulfate, magnesium oxide, magnesium silicate, magnesium stearate,magnesium trisilicate, magnesium trisilicate anhydrous, malic acid,malt, maltitol, maltitol solution, maltodextrin, maltol, maltose,mannitol, medium chain triglycerides, meglumine, menthol,methylcellulose, methyl methacrylate, methyl oleate, methylparaben,methylparaben potassium, methylparaben sodium, microcrystallinecellulose and carboxymethylcellulose sodium, mineral oil, light mineraloil, mineral oil and lanolin alcohols, oil, olive oil, monoethanolamine,montmorillonite, octyl gallate, oleic acid, palmitic acid, paraffin,peanut oil, petrolatum, petrolatum and lanolin alcohols, pharmaceuticalglaze, phenol, liquified phenol, phenoxyethanol, phenoxypropanol,phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate,phenylmercuric nitrate, polacrilin, polacrilin potassium, poloxamer,polydextrose, polyethylene glycol, polyethylene oxide, polyacrylates,polyethylene-polyoxypropylene-block polymers, polymethacrylates,polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives,polyoxyethylene sorbitol fatty acid esters, polyoxyethylene stearates,polyvinyl alcohol, polyvinyl pyrrolidone, potassium alginate, potassiumbenzoate, potassium bicarbonate, potassium bisulfite, potassiumchloride, potassium citrate, potassium citrate anhydrous, potassiumhydrogen phosphate, potassium metabisulfite, monobasic potassiumphosphate, potassium propionate, potassium sorbate, povidone, propanol,propionic acid, propylene carbonate, propylene glycol, propylene glycolalginate, propyl gallate, propylparaben, propylparaben potassium,propylparaben sodium, protamine sulfate, rapeseed oil, Ringer'ssolution, saccharin, saccharin ammonium, saccharin calcium, saccharinsodium, safflower oil, saponite, serum proteins, sesame oil, colloidalsilica, colloidal silicon dioxide, sodium alginate, sodium ascorbate,sodium benzoate, sodium bicarbonate, sodium bisulfite, sodium chloride,anhydrous sodium citrate, sodium citrate dehydrate, sodium chloride,sodium cyclamate, sodium edentate, sodium dodecyl sulfate, sodium laurylsulfate, sodium metabisulfite, sodium phosphate, dibasic, sodiumphosphate, monobasic, sodium phosphate, tribasic, anhydrous sodiumpropionate, sodium propionate, sodium sorbate, sodium starch glycolate,sodium stearyl fumarate, sodium sulfite, sorbic acid, sorbitan esters(sorbitan fatty esters), sorbitol, sorbitol solution 70%, soybean oil,spermaceti wax, starch, corn starch, potato starch, pregelatinizedstarch, sterilizable maize starch, stearic acid, purified stearic acid,stearyl alcohol, sucrose, sugars, compressible sugar, confectioner'ssugar, sugar spheres, invert sugar, Sugartab, Sunset Yellow FCF,synthetic paraffin, talc, tartaric acid, tartrazine, tetrafluoroethane(HFC), theobroma oil, thimerosal, titanium dioxide, alpha tocopherol,tocopheryl acetate, alpha tocopheryl acid succinate, beta-tocopherol,delta-tocopherol, gamma-tocopherol, tragacanth, triacetin, tributylcitrate, triethanolamine, triethyl citrate, trimethyl-β-cyclodextrin,trimethyltetradecylammonium bromide, tris buffer, trisodium edentate,vanillin, type I hydrogenated vegetable oil, water, soft water, hardwater, carbon dioxide-free water, pyrogen-free water, water forinjection, sterile water for inhalation, sterile water for injection,sterile water for irrigation, waxes, anionic emulsifying wax, carnaubawax, cationic emulsifying wax, cetyl ester wax, microcrystalline wax,nonionic emulsifying wax, suppository wax, white wax, yellow wax, whitepetrolatum, wool fat, xanthan gum, xylitol, zein, zinc propionate, zincsalts, zinc stearate, or any excipient in the Handbook of PharmaceuticalExcipients, Third Edition, A. H. Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety.Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin(Mack Publishing Co., Easton, Pa., 1980), which is incorporated byreference in its entirety for all intended purposes, discloses variouscomponents used in formulating pharmaceutically acceptable compositionsand known techniques for the preparation thereof. Except insofar as anyconventional agent is incompatible with the pharmaceutical compositions,its use in pharmaceutical compositions is contemplated. In exemplaryembodiments, the formulations or pharmaceutical compositions of thepresent disclosure do not comprise one or a combination of the aboveingredients. In exemplary embodiments, the formulations orpharmaceutical compositions of the present disclosure comprises none ofthese ingredients. In exemplary aspects, the pharmaceutical compositionof the present disclosure does not comprise dextran. In exemplaryaspects, the pharmaceutical composition of the present disclosure doesnot comprise calcium chloride.

pH

In exemplary embodiments, the pharmaceutical composition of the presentdisclosure has a physiologically compatible pH. Accordingly, in certainembodiments, the AAV formulations are provided that contain a bufferingagent suitable to maintain the formulation at a neutral pH.

In exemplary aspects, the pH of the pharmaceutical composition is about6.5 to about 9.0, about 6.5 to about 8.0, about 6.9 to about 7.7, orabout 7.0 to about 7.5. In certain embodiments, the pH of theformulation is about 6.5 or about 6.6, about 6.7, about 6.8, about 6.9,about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2,about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about8.9, or about 9.0. In exemplary aspects, the pH of the pharmaceuticalcomposition is about 7.0 or about 7.5. In certain embodiments, the pH ofthe pharmaceutical composition is about 7.0. In certain embodiments, thepH of the pharmaceutical composition is about 7.5.

Osmolality

In exemplary embodiments, the pharmaceutical composition of the presentdisclosure has an osmolality of about 200 to about 400 mOsmol/kg, about250 to about 400 mOsmol/kg, or about 290 to about 390 mOsmol/kg. Incertain embodiments, an AAV formulation provided herein will have anosmolality, for example, of about 200 mOsmol/L, about 210 mOsmol/L,about 220 mOsmol/L, about 230 mOsmol/L, about 240 mOsmol/L, about 250mOsmol/L, about 260 mOsmol/L, about 270 mOsmol/L, about 280 mOsmol/L,about 290 mOsmol/L, about 300 mOsmol/L, about 310 mOsmol/L, about 320mOsmol/L, about 330 mOsmol/L, about 340 mOsmol/L, about 350 mOsmol/L,about 360 mOsmol/L, about 370 mOsmol/L, about 380 mOsmol/L, about 390mOsmol/L, or about 400 mOsmol/L. In exemplary aspects, thepharmaceutical composition of the present disclosure has an osmolalityof about 250 mOsmol/kg to about 400 mOsmol/kg. In exemplary aspects, thepharmaceutical composition of the present disclosure has an osmolalityof about 300±10 mOsmol/kg or about 380±10 mOsmol/kg. In certainembodiments, the pharmaceutical composition of the present disclosurehas an osmolality of about 300±10 mOsmol/kg. In certain embodiments, thepharmaceutical composition of the present disclosure has an osmolalityof about 380±10 mOsmol/kg.

Examples of tonocity agents that may be used in the formulationsprovided herein include, without limitation, sodium chloride, dextrose,sucrose, xylitol, fructose, glycerol, sorbitol, mannitol, trehalose,potassium chloride, mannose, calcium chloride, magnesium chloride, otherinorganic salts, other sugars, other sugar alcohols, and combinationsthereof. In certain embodiments, an AAV formulation may comprise atleast one tonocity agent, or at least two, three, four, five, or moretonocity agents.

AAV

In exemplary embodiments, the pharmaceutical composition of the presentdisclosure comprises AAV. The AAV may be of any AAV serotype. Inexemplary aspects, the AAV is of AAV1 serotype, AAV2 serotype, AAV3serotype, AAV4 serotype, AAVS serotype, AAV6 serotype, AAV7 serotype,AAV8 serotype, AAV9 serotype, or AAV10 serotype. In exemplary aspects,the AAV is of AAV8 serotype.

In certain embodiments of the formulations provided herein, the AAV isan rAAV as described in U.S. Patent Application Publication No.2017/0233455 and provisional application No. 62/509,616, which are bothincorporated herein by reference in their entirety and for all purposes.

In exemplary aspects, the pharmaceutical composition comprises a hightiter AAV product. In exemplary aspects, the pharmaceutical compositioncomprises at least about 10¹⁰ virus particles (vp) or at least about10¹¹ virus particles (vp) or at least about 10¹² virus particles (vp) orat least about 10¹³ virus particles (vp). In exemplary aspects, thepharmaceutical composition comprises at least about 10¹⁴ virus particles(vp) or at least about 10¹⁵ virus particles (vp), e.g., at least about2×10¹⁵ virus particles (vp), at least about 5×10¹⁵ virus particles (vp).The pharmaceutical composition comprises also about 10¹⁰ vector genomes(vg) or at least about 10¹¹ vector genomes (vg) or at least about 10¹²vector genomes (vg) or at least about 10¹³ vector genomes (vg). Inexemplary aspects, the pharmaceutical composition comprises at leastabout 10¹⁴ vector genomes (vg) or at least about 10¹⁵ vector genomes(vg), e.g., at least about 2×10¹⁵ vector genomes (vg), at least about5×10¹⁵ vector genomes (vg).

Manufacture Methods and Uses

Methods of preparing a pharmaceutical composition comprising AAV arefurther provided herein. In exemplary aspects, the method comprisescombining about 5 mM to about 25 mM buffering agent, about 50 mM toabout 150 mM or less than about 100 mM pharmaceutically acceptable salt,about 0.001% (w/v) to about 0.01% (w/v) non-ionic surfactant, about 1%(w/v) to about 10% (w/v) sugar and/or sugar alcohol and AAV, therebyobtaining a pharmaceutical composition comprising AAV. In exemplaryaspects, the method comprises preparing a composition, e.g., an aqueoussolution, comprising about 5 mM to about 25 mM buffering agent, about 50mM to about 150 mM or less than about 100 mM pharmaceutically acceptablesalt, about 0.001% (w/v) to about 0.01% (w/v) non-ionic surfactant,about 1% (w/v) to about 10% (w/v) sugar and/or sugar alcohol and addingAAV to the composition. In exemplary aspects, the method comprisespreparing a composition, e.g., an aqueous solution, comprising about 20mM buffering agent, about 70 mM pharmaceutically acceptable salt, about0.005% (w/v) non-ionic surfactant, and about 5% (w/v) sugar and/or sugaralcohol and adding AAV to the composition. In exemplary aspects, themethod comprises preparing a composition, e.g., an aqueous solution,comprising about 10 mM buffering agent about 100 mM pharmaceuticallyacceptable salt, about 0.005% (w/v) non-ionic surfactant, about 5% (w/v)sugar and/or sugar alcohol, and about 50 mM glycine and adding AAV tothe composition. In exemplary aspects, the method comprises preparing acomposition, e.g., an aqueous solution, comprising about 10 mM bufferingagent about 80 mM pharmaceutically acceptable salt, about 0.005% (w/v)non-ionic surfactant, about 5% (w/v) sugar and/or sugar alcohol, andabout 50 mM glycine and adding AAV to the composition. In exemplaryaspects, the method comprises preparing a composition, e.g., an aqueoussolution, comprising about 10 mM buffering agent about 70 mMpharmaceutically acceptable salt, about 0.005% (w/v) non-ionicsurfactant, about 5% (w/v) sugar and/or sugar alcohol, and about 50 mMglycine and adding AAV to the composition. In exemplary aspects, themethod comprises preparing a composition, e.g., an aqueous solution,comprising about 10 mM buffering agent about 60 mM pharmaceuticallyacceptable salt, about 0.005% (w/v) non-ionic surfactant, about 5% (w/v)sugar and/or sugar alcohol, and about 50 mM glycine and adding AAV tothe composition. In exemplary aspects, the method comprises preparing acomposition, e.g., an aqueous solution, comprising about 10 mM bufferingagent about 50 mM pharmaceutically acceptable salt, about 0.005% (w/v)non-ionic surfactant, about 5% (w/v) sugar and/or sugar alcohol, andabout 50 mM glycine and adding AAV to the composition. In exemplaryaspects, the method comprises preparing a composition, e.g., an aqueoussolution, comprising about 10 mM buffering agent about 40 mMpharmaceutically acceptable salt, about 0.005% (w/v) non-ionicsurfactant, about 5% (w/v) sugar and/or sugar alcohol, and about 50 mMglycine and adding AAV to the composition. In exemplary aspects, themethod comprises preparing a composition, e.g., an aqueous solution,comprising about 10 mM buffering agent about 30 mM pharmaceuticallyacceptable salt, about 0.005% (w/v) non-ionic surfactant, about 5% (w/v)sugar and/or sugar alcohol, and about 50 mM glycine and adding AAV tothe composition. In exemplary aspects, the method comprises preparing acomposition, e.g., an aqueous solution, comprising about 10 mM bufferingagent about 20 mM pharmaceutically acceptable salt, about 0.005% (w/v)non-ionic surfactant, about 5% (w/v) sugar and/or sugar alcohol, andabout 50 mM glycine and adding AAV to the composition. In exemplaryaspects, the method comprises preparing a composition, e.g., an aqueoussolution, comprising about 10 mM buffering agent about 10 mMpharmaceutically acceptable salt, about 0.005% (w/v) non-ionicsurfactant, about 5% (w/v) sugar and/or sugar alcohol, and about 50 mMglycine and adding AAV to the composition. In exemplary aspects, themethod comprises preparing a composition, e.g., an aqueous solution,comprising about 10 mM buffering agent about 0 mM pharmaceuticallyacceptable salt, about 0.005% (w/v) non-ionic surfactant, about 5% (w/v)sugar and/or sugar alcohol, and about 50 mM glycine and adding AAV tothe composition. In exemplary aspects, the method comprises preparing acomposition, e.g., an aqueous solution, comprising about 20 mM bufferingagent, about 60 mM pharmaceutically acceptable salt, about 0.005% (w/v)non-ionic surfactant, about 35 mM sugar and/or sugar alcohol, and about110 mM mannitol and adding AAV to the composition. In certainembodiments, the aqueous solution is lyophilized.

Methods of preparing a pharmaceutical composition comprising AAV arefurther provided herein. In exemplary aspects, the method comprisescombining about 5 mM to about 25 mM L-histidine, about 50 mM to about150 mM sodium chloride, about 0.001% (w/v) to about 0.01% (w/v)polysorbate 80 (PS80), about 1% (w/v) to about 10% (w/v) sucrose ortrehalose and AAV, thereby obtaining a pharmaceutical compositioncomprising AAV. In exemplary aspects, the method comprises preparing acomposition, e.g., an aqueous solution, comprising about 5 mM to about25 mM L-histidine, about 50 mM to about 150 mM sodium chloride, about0.001% (w/v) to about 0.01% (w/v) polysorbate 80 (PS80), and about 1%(w/v) to about 10% (w/v) sucrose or trehalose and adding AAV to thecomposition. In exemplary aspects, the method comprises preparing acomposition, e.g., an aqueous solution, comprising about 20 mML-histidine, about 70 mM sodium chloride, about 0.005% (w/v) polysorbate80 (PS80), and about 5% (w/v) sucrose and adding AAV to the composition.In exemplary aspects, the method comprises preparing a composition,e.g., an aqueous solution, comprising about 10 mM L-histidine, about 100mM sodium chloride, about 0.005% (w/v) polysorbate 80 (PS80), about 5%(w/v) trehalose, and about 50 mM glycine and adding AAV to thecomposition. In exemplary aspects, the method comprises preparing acomposition, e.g., an aqueous solution, comprising about 10 mML-histidine, about 80 mM sodium chloride, about 0.005% (w/v) polysorbate80 (PS80), about 5% (w/v) trehalose, and about 50 mM glycine and addingAAV to the composition. In exemplary aspects, the method comprisespreparing a composition, e.g., an aqueous solution, comprising about 10mM L-histidine, about 70 mM sodium chloride, about 0.005% (w/v)polysorbate 80 (PS80), about 5% (w/v) trehalose, and about 50 mM glycineand adding AAV to the composition. In exemplary aspects, the methodcomprises preparing a composition, e.g., an aqueous solution, comprisingabout 10 mM L-histidine, about 60 mM sodium chloride, about 0.005% (w/v)polysorbate 80 (PS80), about 5% (w/v) trehalose, and about 50 mM glycineand adding AAV to the composition. In exemplary aspects, the methodcomprises preparing a composition, e.g., an aqueous solution, comprisingabout 10 mM L-histidine, about 50 mM sodium chloride, about 0.005% (w/v)polysorbate 80 (PS80), about 5% (w/v) trehalose, and about 50 mM glycineand adding AAV to the composition. In exemplary aspects, the methodcomprises preparing a composition, e.g., an aqueous solution, comprisingabout 10 mM L-histidine, about 40 mM sodium chloride, about 0.005% (w/v)polysorbate 80 (PS80), about 5% (w/v) trehalose, and about 50 mM glycineand adding AAV to the composition. In exemplary aspects, the methodcomprises preparing a composition, e.g., an aqueous solution, comprisingabout 10 mM L-histidine, about 30 mM sodium chloride, about 0.005% (w/v)polysorbate 80 (PS80), about 5% (w/v) trehalose, and about 50 mM glycineand adding AAV to the composition. In exemplary aspects, the methodcomprises preparing a composition, e.g., an aqueous solution, comprisingabout 10 mM L-histidine, about 20 mM sodium chloride, about 0.005% (w/v)polysorbate 80 (PS80), about 5% (w/v) trehalose, and about 50 mM glycineand adding AAV to the composition. In exemplary aspects, the methodcomprises preparing a composition, e.g., an aqueous solution, comprisingabout 10 mM L-histidine, about 10 mM sodium chloride, about 0.005% (w/v)polysorbate 80 (PS80), about 5% (w/v) trehalose, and about 50 mM glycineand adding AAV to the composition. In exemplary aspects, the methodcomprises preparing a composition, e.g., an aqueous solution, comprisingabout 10 mM L-histidine, about 0 mM sodium chloride, about 0.005% (w/v)polysorbate 80 (PS80), about 5% (w/v) trehalose, and about 50 mM glycineand adding AAV to the composition. In exemplary aspects, the methodcomprises preparing a composition, e.g., an aqueous solution, comprisingabout 20 mM L-histidine, about 60 mM sodium chloride, about 0.005% (w/v)polysorbate 80 (PS80), about 35 mM trehalose, and about 110 mM mannitoland adding AAV to the composition. In certain embodiments, the aqueoussolution is lyophilized.

In certain embodiments, lyophilization is used to produce a dehydratedcomposition, and thus the composition (e.g., the composition thatcontains the AAV) is a lyophilized composition. In some instances, alyophilized composition is a composition where water has been removedfrom the composition by sublimation, where the water in the compositionundergoes a phase transition from a solid to a gas. For example, alyophilized composition may be a composition where water has beenremoved from the composition by freezing the composition (e.g., freezingthe water in the composition) and then reducing the pressure surroundingthe composition such that the water in the composition undergoessublimation. As described above, a lyophilized composition may includewater in a low amount, such as 25% or less, or 20% or less, or 15% orless, or 10% or less, or 9% or less, or 8% or less, or 7% or less, or 6%or less, or 5% or less, or 4% or less, or 3% or less, or 2% or less, or1° A or less, or 0.5% or less, or 0.25% or less, or 0.1° A or less wateras measured by Karl Fischer (KF) titration. In certain embodiments, alyophilized composition has 3% or less water as measured by Karl Fischertitration. In certain embodiments, a lyophilized composition has 2% orless water as measured by Karl Fischer titration. In certainembodiments, a lyophilized composition has 1° A or less water asmeasured by Karl Fischer titration. In certain embodiments, alyophilized composition has 0.5% or less water as measured by KarlFischer titration.

During lyophilization, the temperature of the composition may bereduced, for example to a temperature below the freezing point of waterin the composition. For example, the temperature of the composition maybe reduced to 0° C. or less, or −5° C. or less, or −10° C. or less, or−15° C. or less, or −20° C. or less, or −25° C. or less, or 30° C. orless, or −35° C. or less, or −40° C. or less, or −45° C. or less, or−50° C. or less, or 55° C. or less, or −60° C. or less, or −65° C. orless, or −75° C. or less. In some cases, the temperature of thecomposition is reduced to −20° C. In some cases, the temperature of thecomposition is reduced to −60° C. (set point −80° C.).

In certain embodiments, the pressure surrounding the composition isreduced below standard atmospheric pressure. For example, the pressuresurrounding the composition may be reduced to 500 Torr or less, such as250 Torr or less, or 100 Torr or less, or 50 Torr or less, or 10 Torr orless, or 1 Torr or less, or 500 mTorr or less, or 400 mTorr or less, or300 mTorr or less, or 200 mTorr or less, or 100 mTorr or less, or 90mTorr or less, or 80 mTorr or less, or 70 mTorr or less, or 60 mTorr orless, or 50 mTorr or less, or 40 mTorr or less, or 30 mTorr or less, or20 mTorr or less, or 10 mTorr or less, or 5 mTorr or less, or 1 mTorr orless. In some cases, the pressure surrounding the composition is reducedto 40 to 50 mTorr or less, and such as 1.5 to 2 mTorr.

In some embodiments, lyophilizing may also include increasing thetemperature of the composition while the pressure surrounding thecomposition is reduced. For example, the temperature of the compositionmay be increased from a minimum temperature as described above to atemperature greater than the minimum temperature. In some cases, thetemperature is increased to facilitate sublimation of the water in thecomposition at the reduced surrounding pressure.

Embodiments of the method of making the lyophilized AAV composition mayalso include producing the aqueous concentrate composition, which issubsequently lyophilized.

In exemplary aspects, the method comprises placing the compositioncomprising AAV in a glass or plastic container, e.g., a glass vial, or aplastic tube. In exemplary aspects, the glass or plastic container isany one of those known in the art, commercially available, and/ordescribed herein (see, e.g., Example 3). In exemplary aspects, themethod comprises placing about 0.1 mL to about 10 ml of the compositioncomprising AAV into the glass or plastic container. In exemplaryaspects, the method comprises placing about 0.2 mL to about 6 ml (e.g.,about 0.2 ml, about 0.3 ml, about 0.5 mol, about 5.6 ml) of thecomposition comprising AAV into the glass or plastic container. Methodsof preparing pharmaceutical compositions are described herein (see,e.g., Examples 1 and 2).

Providing the composition in a container may facilitate maintaining thecomposition as a sterile composition. For instance, the container may beconfigured to maintain the composition enclosed in the container in asterile environment. As such, the container may be a sealed container,for example the container may include a seal, such as a water-tightand/or an air-tight seal. The seal may be removable from the containerto allow a user access to the contents of the container. In someinstances, the seal may be a frangible seal, or in other instances, theseal may be configured to allow insertion of a needle, cannula orsyringe into the interior of the container without removing the sealfrom the container. In some cases, a seal configured to allow access tothe interior of the container without removing the seal from thecontainer may facilitate maintaining the contents of the container(e.g., the composition in the container) in a sterile environment priorto administration of the composition to a subject. Suitable materialsfor the seal include, for example, rubber or polymer seals, such as, butnot limited to, silicone rubber, natural rubber, styrene butadienerubber, ethylene-propylene copolymers, polychloroprene, polyacrylate,polybutadiene, polyurethane, styrene butadiene, and the like, andcombinations thereof. For example, in certain embodiments, the seal is aseptum pierceable by a needle, syringe, or cannula. The seal may alsoprovide convenient access to a sample in the container, as well as aprotective barrier that overlies the opening of the container. In someinstances, the seal is a removable seal, such as a threaded or snap-oncap or other suitable sealing element that can be applied to the openingof the container. For instance, a threaded cap can be screwed over theopening before or after a sample has been added to the container.

In some cases, the container is a unit dosage container. A unit dosagecontainer refers to a container that contains one or more unitarydosages for administration to a subject. In some embodiments, a unitdosage container includes a predetermined quantity of a subjectcomposition calculated in an amount sufficient to produce a desiredeffect in a subject. Certain embodiments of the compositions may beprovided in a unit dosage container suitable for individualadministration of precise dosages. The amount of active compositionadministered to a subject may depend on the subject being treated, theseverity of the affliction, and the manner of administration. Forexample, the unit dosage container may contain a quantity of thecomposition to be administered as disclosed herein in an amounteffective to achieve the desired effect in the subject being treated. Incertain instances, a unit dosage container includes a composition havinga AAV in a therapeutically effective amount. In certain embodiments, theunit dosage container is a vial. In some cases, the vial is a sealedvial (e.g., as described above regarding a sealed container).

The container may be composed of any convenient material that iscompatible with the AAV and other components of the composition. Forexample, the container can be a solid-compatible container configured tocontain a solid (e.g., a lyophilized composition). In some instances,the container is a liquid-compatible container configured to contain aliquid. Containers may also be solid and liquid compatible, where thecontainer is configured to contain solids and liquids. In some cases, aliquid in the container may be an aqueous liquid, and in these cases,the container may be compatible with aqueous compositions. By“compatible” is meant that the container is substantially inert (e.g.,does not significantly react with) the liquid and/or compositions orother components in contact with the container. Examples of suitablecontainer materials include, but are not limited to, glass and plastic.For example, the container may be composed of glass, such as, but notlimited to, silicate glass, borosilicate glass, sodium borosilicateglass (e.g., PYREX™), fused quartz glass, fused silica glass, and thelike. Other examples of suitable container materials for the containerinclude plastics, such as, but not limited to, polypropylene,polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroethers(PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxy alkanes(PFA), polyethylene terephthalate (PET), polyethylene (PE),polyetheretherketone (PEEK), polystyrene, and the like. In certaininstances, as described above, the container is a vial, and as such maybe a glass vial. As described above, the container may be a sealedcontainer, and as such may be a sealed glass vial.

In exemplary aspects, the volume of the composition comprising AAVplaced into the glass or plastic container is about 0.1 ml to about 10ml, about 0.1 ml to about 5 ml, about 0.2 ml to about 6 ml, about 0.2 mlto about 5 ml, about 0.25 ml to about 5 ml, about 2 ml to about 5 ml,about 2.5 ml to about 5 ml, about 3 ml to about 5 ml, about 4 ml toabout 5 ml, about 5 ml to about 10 ml, about 6 ml about 9 ml or about 7ml to about 8 ml. In exemplary aspects, the volume about 0.1 ml about0.2 ml, about 0.25 ml, about 0.3 ml, about 0.4 about 0.5 ml, about 0.6ml, about 0.7 ml. about 0.75 ml, about 0.8 ml, about 0.9 ml, about 1 ml,about 2 ml, about 2.5 ml, about 3 ml, about 4 ml, about 5 ml, about 5.5ml, about 5.6 ml, about 6 ml, about 7 ml, about 7.5 ml, about 8 ml,about 9 ml, or about 10 ml.

In exemplary aspects, the composition comprising AAV is stored at atemperature of about −80° C. to about −10° C. for at least 1 month(e.g., at least 2 months, at least 3 months, at least 4 months, at least5 months, at least 6 months). In exemplary aspects, the compositioncomprising AAV is stored at a temperature of about −60° C. to about −20°C. for at least 1 month (e.g., at least 2 months, at least 3 months, atleast 4 months, at least 5 months, at least 6 months). In exemplaryaspects, the composition comprising AAV is stored for 3, 4, 5, or 6months or longer. Methods of storing the composition are describedherein (see, e.g., Examples 1 to 3). In exemplary aspects, greater than80% of the initial amount of AAV (e.g., the amount of AAV in thecomposition prior to storage) is potent after the storage period (e.g.,a storage period of about 3 months, about 4 months, about 5 months, orabout 6 months or longer). In exemplary aspects, greater than 90% of theinitial amount of AAV is potent after the storage period (e.g., astorage period of about 3 months, about 4 months, about 5 months, orabout 6 months or longer). In exemplary aspects, greater than 95% of theinitial amount of AAV is potent after the storage period (e.g., astorage period of about 3 months, about 4 months, about 5 months, orabout 6 months or longer). In exemplary aspects, the biopotency of theAAV at the end of the storage period is substantially the same as thebiopotency of the AAV at the beginning of the storage period. Inexemplary aspects, the biopotency of the AAV at the end of the storageperiod is increased relative to the biopotency of the AAV at thebeginning of the storage period. In exemplary aspects, the appearance ofthe composition at the end of the storage period is substantially thesame as the composition at the beginning of the storage period. Inexemplary aspects, the appearance of the composition at the end of thestorage period is characterized by having no visible particles. Inexemplary aspects, the particle concentration of the composition at theend of the storage period is substantially the same as the particleconcentration of the composition at the beginning of the storage period.In exemplary aspects, the particle concentration of the composition atthe end of the storage period is determined by microflow imaging (MFI).

Methods of treating a subject for a disorder treatable by gene therapyare provided by the present disclosure. In exemplary aspects, the methodcomprises administering to the subject a pharmaceutical composition asdescribed herein in an amount effective to treat the disorder. Inexemplary aspects, the disorder is a bleeding disorder (e.g., hemophiliaA or B) and the method comprises administering to the subject apharmaceutical composition of the present disclosure in an amounteffective to treat the bleeding disorder.

In exemplary aspects, the methods comprise administering thepharmaceutical composition by parenteral administration. The term,“parenteral” means not through the alimentary canal but by some otherroute. For example, the formulations disclosed herein may be formulatedfor administration via known methods, such as intravenous administration(e.g., as a bolus or by continuous infusion over a period of time), byintramuscular, intraperitoneal, intracerobrospinal, subcutaneous,intra-articular, intrasynovial, intrathecal, oral, topical, orinhalation routes. In certain embodiments, the AAV formulations providedherein can be administered either systemically or locally. Systemicadministration includes, without limitation: oral, subdermal,intraperitoneal, subcutaneous, transnasal, sublingual, or rectal routesof administration. Local administration includes, without limitation:topical, subcutaneous, intramuscular, and intraperitoneal routes ofadministration

Methods of administering AAV for gene therapy of a subject are known inthe art. See, e.g., Monahan, Gene therapy in an era of emergingtreatment options for hemophilia B, J Thromb Haemost. 2015 June; 13(01): S151-S160.

Reconstituted compositions of the present disclosure may be administeredto a subject, for example by injection or intravenously. In theseembodiments, prior to administration of the reconstituted composition toa subject, a solid composition, e.g., as described above, may becombined with a liquid to provide a liquid composition suitable foradministration, for example by injection or intravenously. In somecases, prior to administration of the composition to a subject, a solidcomposition may be combined with water (e.g., water for injection, WFI)or buffer/buffering agent (e.g., as described above) to provide anaqueous composition suitable for administration, for example byinjection or intravenously. For instance, a lyophilized composition maybe reconstituted with water (e.g., water for injection, WFI) orbuffer/buffering agent (e.g., as described above) to produce areconstituted dosage unit suitable for administration to a subject, forexample by injection or intravenously.

In certain embodiments, the reconstituted dosage unit has a pHcompatible with physiological conditions. In some cases, the pH of thereconstituted dosage unit ranges from 6 to 8. In some cases, the pH ofthe reconstituted dosage unit ranges from 7 to 8. For example, the pH ofthe reconstituted dosage unit may range from 7 to 7.5. In some cases,the pH of the reconstituted dosage unit is 7.0. In some cases, the pH ofthe reconstituted dosage unit is 7.1. In some cases, the pH of thereconstituted dosage unit is 7.2. In some cases, the pH of thereconstituted dosage unit is 7.3. In some cases, the pH of thereconstituted dosage unit is 7.4.

The reconstituted dosage unit may include a predetermined quantity ofthe composition of the present disclosure calculated in an amountsufficient to produce a desired therapeutic effect in a subject. Theamount of the composition in a reconstituted dosage unit that isadministered to a subject may depend on the subject being treated, theseverity of the affliction, and the manner of administration. Forexample, the reconstituted dosage unit may include a quantity of thecomposition to be administered as disclosed herein in a therapeuticallyeffective amount.

When administered to a subject, the liquid or reconstituted dosage unitmay include a therapeutically effective amount of the AAV such that thereconstituted dosage unit delivers from 1E+10 vp/ml to 5E+15 vp/ml or1E+10 cp/ml to 5E+15 cp/ml.

In certain embodiments, the method includes administering the liquid orreconstituted dosage unit to the subject according to a treatmentregimen. For example, in some cases, a subject to be treated may havebeen prescribed a treatment regimen from a health care provider. In somecases, a treatment regimen includes, but is not necessarily limited to,administration five times per day, four times per day, three times perday, twice per day, once per day, three times per week, twice per week,once per week, once every two weeks, once every three weeks, once permonth, once every 5 weeks, once every 6 weeks, once every 7 weeks, onceevery other month, and any combination thereof.

In some embodiments, the treatment regimen includes administering one ormore doses over an extended period of time. In certain cases, a singledose (e.g., a single dosage unit) is administered to the subject, andthe initial dose may be followed by one or more doses administered tothe subject at a subsequent time. In some instances, more than one dose(e.g., more than one dosage unit) is administered to the subject, andthe initial doses may be followed by one or more doses administered tothe subject at a subsequent time. For example, a single dose (e.g., asingle dosage unit) may be administered to the subject, and the singledose may be followed by a single dose administered to the subject at asubsequent time. Additional single doses may be administered atsubsequent points in time. In other cases, a single dose (e.g., a singledosage unit) may be administered to the subject, and the single dose maybe followed by two doses administered to the subject at a subsequenttime. Additional single or multiple doses may be administered atsubsequent points in time.

In certain embodiments, reconstituted dosage units of the presentdisclosure can be administered prior to, concurrent with, or subsequentto other active agents for treating related or unrelated conditions,e.g., in combination therapy. Examples of such additional therapiesinclude radiation therapies, surgical therapies and chemotherapeutictherapies. If provided at the same time as other active agents,reconstituted dosage units of the present disclosure can be provided inthe same or in a different formulation. For example, concurrent therapymay be achieved by administering a reconstituted dosage unit and apharmaceutical composition having at least one other active agent, suchas a chemotherapeutic agent, which in combination provide atherapeutically effective dose, according to a particular treatmentregimen. Administration of separate pharmaceutical compositions can beperformed simultaneously or at different times (e.g., sequentially, ineither order, on the same day, or on different days), as long as atherapeutically effective effect of the combination of these substancesis caused in the subject undergoing therapy.

Accordingly, aspects of the present disclosure further includecombination therapies. In certain embodiments, the subject methodincludes administering a therapeutically effective amount of one or moreadditional active agents. By combination therapy is meant that a AAVcomposition (e.g., as described herein) can be used in a combinationwith another therapeutic agent to treat a single disease or condition.In certain embodiments, a compound of the present disclosure isadministered concurrently with the administration of another therapeuticagent, which can be administered as a component of a compositionincluding the compound of the present disclosure or as a component of adifferent composition. In certain embodiments, a composition including acompound of the present disclosure is administered prior or subsequentto administration of another therapeutic agent.

The following examples are given merely to illustrate the presentinvention and not in any way to limit its scope.

EXAMPLES Example 1

This example demonstrates initial studies that led to the selection ofthree formulations for further development.

Six different formulations were manufactured. Briefly, a single lotcomprising mostly empty capsid material was diluted withPBS/NaCl/Sorbitol buffer to generate 180.8 g. The solution was spikedwith 0.005% Croda super refined Tween 80 to prevent excessive materialloss via adsorption. This solution was divided into 6 parts, eachcontaining 30 g material. The buffer solution was then changed into oneof the six buffers using Slide-A-Lyzer dialysis cassettes and this stepwas performed three times with 100-fold volume. After the three dialysissteps, each of the dialyzed solutions were filled into 2-ml SiO₂ vials(filling volume=0.25 ml) and then stored at −20±2° C. and ≤−60° C. (setpoint: −80° C.) for up to 6 months and at 5±3° C. for up to 4 months.Tables 1-6 describe Formulations 1-6 (also referred to as Buffers 1-6,and Samples 1-6), respectively.

TABLE 1 Formulation 1: PBS/NaCl/Sorbitol buffer with Polysorbate 80(PS80) Concentration Concentration Excipient [mM] g/kg buffer Sodiumchloride 350 20.45 Potassium chloride 2.68 0.20Disodiumhydrogenphosphate- 8.09 1.44 Dihydrate Monopotassium phosphate1.47 0.20 D-Sorbitol    5% 50.00 Croda super refined Tween 80 (PS80)0.005% 0.05 25% HCl pH adjustment 1M NaOH Purified water add to 1 kg pHvalue 7.4 ± 0.3

TABLE 2 Formulation 2 Concentration Concentration Excipient [mM] g/kgbuffer L-histidine 20 3.103 NaCl 70 4.091 Sucrose    5% 50.00 Crodasuper refined Tween 80 0.005% 0.050 (PS80) Purified water add to 1 kg pHvalue 7.5

TABLE 3 Formulation 3 Concentration Concentration Excipient [mM] g/kgbuffer L-histidine 20 3.103 NaCl 60 3.506 Mannitol 110 20.039 TrehaloseDihydrate 35 13.24 Croda super refined Tween 80 (PS80) 0.005% 0.05Purified water add to 1 kg pH value 7.0 ± 0.1

TABLE 4 Formulation 4 Concentration Concentration Excipient [mM] g/kgbuffer L-histidine 10 1.552 NaCl 100 5.844 Glycine 50 3.754 TrehaloseDihydrate    5% 50.00 Croda super refined Tween 80 0.005% 0.05 Purifiedwater add to 1 kg pH value 7.0 ± 0.1

TABLE 5 Formulation 5 Concentration Concentration Excipient [mM] g/kgbuffer Tri-Sodium Citrate-di-Hydrate 15 4.41 Glycine 15 1.11 Mannitol 2%20.00 Trehalose Dihydrate 1% 10.00 Croda super refilled Tween 80 (PS80)0.005%    0.05 Purified water add to 1 kg pH value 7.3 ± 0.1

TABLE 6 Formulation 6 Concentration Concentration Excipient [mM] g/kgbuffer TRIS 80 9.691 Dextran T40 1.77% 17.70 Dextran T10 0.53% 5.30Sucrose  6.9% 69.00 Croda super refined Tween 80 (PS80) 0.005%  0.05Purified water add to 1 kg pH value 7.1

The total particle titer (empty+full) was tested by a total rAAVparticle ELISA. AAV purity and chemical degradation was tested bySDS-PAGE (fluorescence staining, 4-12% BisTris gel with Flamingo Stain).Physical degradation (damaged capsids or fragments) were tested by SECand MFI. The appearance and pH were also tested.

The formulations stored at −20±2° C. and −60° C. (set point: −80° C.)were tested for MFI, appearance, pH, total rAAV8 particle ELISA,SDS-PAGE, and SEC at the test time points indicated in Table 7. An “-”indicates no samples were taken.

TABLE 7 Study outline for samples stored at −20 ± 2° C. and ≤−60° C.(*set point −80° C.) TOTAL Test Time Point (months) Number Sample 2 ofTest Volume 0 1 8 weeks 3 6 Samples MFI   3 ml x — — — x 50 Appearance  1 ml x x x x x 10 pH value x x x x x Total rAAV8 particle 2× x x x x x100 ELISA 0.3 ml SDS-PAGE 0.3 ml x x x x x 50 (fluorescence staining,4-12% BisTris gel with Flamingo Stain) SEC 0.3 ml x x x x x 50

The formulations stored at 5±3° C. were tested for MFI, appearance, pH,total rAAV8 particle ELISA, SDS-PAGE, and SEC at the test time pointsindicated in Table 8. An “-” indicates no samples were taken.

TABLE 8 Study outline for samples stored at +5 ± 3° C. Test time point(months) Total Sample 2 Number of Test Volume 0 1 (8 weeks) 4 SamplesMFI   3 ml x — — — x Appearance   1 ml x x x x 20 pH value x x x x TotalrAAV8 particle 2x x x x x 40 ELISA 0.3 ml SDS-PAGE (fluorescence 0.3 mlx x x x 20 staining, 4-12% BisTris gel with Flamingo Stain) SEC 0.3 ml xx x x 20

Results of the appearance test are shown in Table 9, wherein theclassification of particles inspection with an unaided eye were asfollows: Level A=no particles visible; Level B=small single particles,barely visible; Level C=small single particles, easily visible; Level Dmany small particles, easily visible; and Level E=particles visiblegreater than or equal to 1 mm.

TABLE 9 Appearance with storage at +5 ± 3° C., −20 ± 2° C., and ≤−60° C.testing time point/buffer at +5 ± 3° C. 0 months 1 month 8 weeks 4months buffer 1 at +5 ± 3° C. A A-B A-B C buffer 2 at +5 ± 3° C. A A A Abuffer 3 at +5 ± 3° C. A A A A buffer 4 at +5 ± 3° C. A A A A buffer 5at +5 ± 3° C. A A A A buffer 6 at +5 ± 3° C. A (yellow) A (yellow) A(yellow) A (yellow) 0 months 1 month 8 weeks 3 months 6 months testingtime point/buffer at −20 ± 2° C. buffer 1 at −20 ± 2° C. A A-B A-B A-BA-B buffer 2 at −20 ± 2° C. A A A A A buffer 3 at −20 ± 2° C. A A A A Abuffer 4 at −20 ± 2° C. A A A A A buffer 5 at −20 ± 2° C. A A A A Abuffer 6 at −20 ± 2° C. A (yellow) A (yellow) A (yellow) A (yellow) A(yellow) testing time point/buffer at ≤−60° C. buffer 1 at ≤−60° C. AA-B A-B A A buffer 2 at ≤−60° C. A A A A A buffer 3 at ≤−60° C. A A A AA buffer 4 at ≤−60° C. A A A A A buffer 5 at ≤−60° C. A A A A A buffer 6at ≤−60° C. A (yellow) A (yellow) A (yellow) A (yellow) A (yellow)

Results of the pH assays are shown in Table 10. No significant shiftscan be seen for the pH values after being stored at 5±3° C. for up to 4months and at −20±2° C. and −60° C. for up to 6 months after thawingthem.

TABLE 10 Total pH values with storage at +5 ± 3° C., −20 ± 2° C., and≤−60° C. target testing time point/buffer at +5 ± 3° C. pH value 0months 1 month 8 weeks 4 months buffer 1 at +5 ± 3° C. 7.4 7.24 7.297.31 7.31 buffer 2 at +5 ± 3° C. 7.5 7.37 7.38 7.40 7.47 buffer 3 at +5± 3° C. 7.0 6.86 6.95 7.00 7.01 buffer 4 at +5 ± 3° C. 7.0 6.93 6.897.01 7.01 buffer 5 at +5 ± 3° C. 7.3 7.22 7.08 7.13 7.23 buffer 6 at +5± 3° C. 7.1 7.12 7.11 7.10 7.09 testing time point/ target buffer at −20± 2° C. pH value 0 months 1 month 8 weeks 3 months 6 months buffet 1 at−20 ± 2° C. 7.4 7.24 7.32 7.33 7.34 7.33 buffer 2 at −20 ± 2° C. 7.57.37 7.37 7.42 7.40 7.38 buffer 3 at −20 ± 2° C. 7.0 6.86 7.00 7.02 7.007.00 buffer 4 at −20 ± 2° C. 7.0 6.93 6.97 6.99 6.97 6.99 buffer 5 at−20 ± 2° C. 7.3 7.22 6.90 6.93 6.92 6.93 buffer 6 at −20 ± 2° C. 7.17.12 6.88 6.98 6.98 6.98 target testing time point/buffer at ≤−60° C. pHvalue 0 months 1 month 8 weeks 3 months 6 months buffer 1 at ≤−60° C.7.4 7.24 7.31 7.34 7.34 7.31 buffer 2 at ≤−60° C. 7.5 7.37 7.40 7.427.41 7.39 buffer 3 at ≤−60° C. 7.0 6.86 6.98 7.04 7.01 7.00 buffer 4 at≤−60° C. 7.0 6.93 6.89 7.00 7.00 7.00 buffer 5 at ≤−60° C. 7.3 7.22 6.886.93 6.94 6.92 buffer 6 at ≤−60° C. 7.1 7.12 6.86 6.98 6.98 6.97

Results of the AAV8 titration ELISA are shown in Table 11. All resultsduring storage at 5±3° C., −20±2° C., and −60° C. do not show anysignificant differences among the six formulations investigated.Harmonized results (calculated with 0.44) are shown.

TABLE 11 Calculated AAV8 Titration ELISA with storage at +5 ± 3° C., −20± 2° C., and ≤−60° C. testing time point/buffer at +5 ± 3° C. 0 months 1month 8 weeks 4 months buffer 1 at +5 ± 3° C. 1.45E+13 1.74E+13 2.43E+131.77E+13 buffer 2 at +5 ± 3° C. 1.45E+13 1.57E+13 1.93E+13 1.80E+13buffer 3 at +5 ± 3° C. 1.31E+13 1.49E+13 1.94E+13 1.64E+13 buffer 4 at+5 ± 3° C. 1.46E+13 1.57E+13 2.00E+13 1.92E+13 buffer 5 at +5 ± 3° C.1.41E+13 1.52E+13 2.02E+13 1.72E+13 buffer 6 at +5 ± 3° C. 1.74E+131.88E+13 2.42E+13 2.09E+13 0 months 1 month 8 weeks 3 months 6 monthstesting time point/buffer at −20 ± 2° C. buffer 1 at −20 ± 2° C.1.45E+13 1.58E+13 1.93E+13 1.89E+13 1.92E+13 buffer 2 at −20 ± 2° C.1.45E+13 1.59E+13 1.83E+13 1.87E+13 1.89E+13 buffer 3 at −20 ± 2° C.1.31E+13 1.48E+13 1.70E+13 1.74E+13 1.71E+13 buffer 4 at −20 ± 2° C.1.46E+13 1.57E+13 1.82E+13 1.86E+13 1.95E+13 buffer 5 at −20 ± 2° C.1.41E+13 1.47E+13 1.78E+13 1.78E+13 1.81E+13 buffer 6 at −20 ± 2° C.1.74E+13 1.89E+13 2.22E+13 2.29E+13 2.19E+13 testing time point/bufferat ≤−60° C. buffer 1 at ≤−60° C. 1.45E+13 1.58E+13 2.03E+13 1.80E+131.72E+13 buffer 2 at ≤−60° C. 1.45E+13 1.57E+13 2.19E+13 1.86E+131.72E+13 buffer 3 at ≤−60° C. 1.31E+13 1.50E+13 1.76E+13 1.76E+131.73E+13 buffer 4 at ≤−60° C. 1.46E+13 1.65E+13 1.94E+13 1.78E+131.43E+13 buffer 5 at ≤−60° C. 1.41E+13 1.51E+13 1.82E+13 1.80E+131.64E+13 buffer 6 at ≤−60° C. 1.74E+13 1.98E+13 2.44E+13 2.13E+131.54E+13

Percent aggregates were measured as shown in Table 12. Higher aggregatevalues were seed for Formulation 3 at 0 months and after storage at 5±3°C. and at −60° C. All other buffers show fluctuating values.

TABLE 12 Aggregates [%] with storage at +5 ± 3° C., −20 ± 2° C., and≤−60° C. testing time point/buffer at +5 ± 3° C. 0 months 1 month 8weeks 4 months buffer 1 at +5 ± 3° C. 1.55 1.50 1.90 1.84 buffer 2 at +5± 3° C. 1.59 1.50 1.57 1.85 buffer 3 at +5 ± 3° C. 2.34 1.75 2.00 1.93buffer 4 at +5 ± 3° C. 1.56 1.57 1.50 1.69 buffer 5 at +5 ± 3° C. 1.721.81 1.49 1.81 buffer 6 at +5 ± 3° C. 1.64 1.76 1.88 1.85 0 1 8 3 6months month weeks months months testing time point/buffer at −20 ± 2°C. buffer 1 at −20 ± 2° C. 1.55 1.47 2.21 1.31 1.41 buffer 2 at −20 ± 2°C. 1.59 1.52 2.24 1.43 1.42 buffer 3 at −20 ± 2° C. 2.34 1.49 1.74 1.371.44 buffer 4 at −20 ± 2° C. 1.56 1.58 1.53 1.38 1.50 buffer 5 at −20 ±2° C. 1.72 1.70 1.43 1.90 1.54 buffer 6 at −20 ± 2° C. 1.64 1.69 2.782.41 1.87 testing time point/buffer at ≤−60° C. buffer 1 at ≤−60° C.1.55 1.54 2.30 1.38 1.47 buffer 2 at ≤−60° C. 1.59 1.53 1.70 1.46 1.51buffer 3 at ≤−60° C. 2.34 2.25 1.95 1.61 2.27 buffer 4 at ≤−60° C. 1.561.52 1.63 1.49 1.46 buffer 5 at ≤−60° C. 1.72 2.81 2.07 2.26 1.95 buffer6 at ≤−60° C. 1.64 1.58 1.93 1.47 1.59

The results of the SDS-PAGE assays are shown in Tables 13-18, showingmeasurements at 0, 1, 2, 3, 4, and 6 months. All samples show stabilityduring storage time for all temperatures except Buffer 1, that shows anadditional band at 163 kDa after 4 months' storage at 5±3° C.

TABLE 13 Purity and summary with storage time = 0 months Sample 1 Sample2 Sample 3 Sample 4 Sample 5 Sample 6 Band Band % Band % Band % Band %Band % Band % VP1 15.85 15.33 16.11 16.58 16.62 16.43 VP2 22.55 22.9222.08 23.58 23.4 24.51 VP3 61.6 61.75 61.82 59.84 59.98 59.06 SUM VP1 +VP2 + VP3 100.0 100.0 100.0 100.0 100.0 100.0

TABLE 14 Purity and summary with storage time = 1 month at +5 ± 3° C.Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 lane 3 lane 4 lane5 lane 6 lane 7 lane 8 Band Band % Band % Band % Band % Band % Band %VP1 15.8 16.7 16.5 16.6 16.6 17.3 VP2 27.1 27.1 26.3 26.7 26.2 26.7 VP357.2 56.2 57.3 56.6 57.2 56.0 SUM VP1 + VP2 + VP3 100.0 100.0 100.0100.0 100.0 100.0 Purity and summary with storage time = 1 month at −20± 2° C. Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 lane 9lane 10 lane 11 lane 12 lane 13 lane 14 Band Band % Band % Band % Band %Band % Band % VP1 16.8 16.7 16.3 17.1 16.6 17.0 VP2 26.1 26.2 26.2 26.225.4 26.3 VP3 57.2 57.1 57.5 56.8 58.1 56.7 SUM VP1 + VP2 + VP3 100.0100.0 100.0 100.0 100.0 100.0 Purity and summary with storage time = 1month at ≤−60° C. Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6lane 15 lane 16 lane 17 lane 18 lane 19 lane 20 Band Band % Band % Band% Band % Band % Band % VP1 16.5 16.5 16.3 16.4 16.4 17.7 VP2 26.3 26.826.1 26.9 26.5 27.2 VP3 57.2 56.7 57.6 56.7 57.1 55.1 SUM VP1 + VP2 +VP3 100.0 100.0 100.0 100.0 100.0 100.0

TABLE 15 Purity and summary with storage time = 2 months at +5 ± 3° C.Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Lane 3 Lane 4 Lane5 Lane 6 Lane 7 Lane 8 Band Band % Band % Band % Band % Band % Band %VP1 15.3 16.2 16.3 16.3 16.6 16.9 VP2 26.6 26.5 26.4 26.4 26.5 26.4 VP358.1 57.3 57.3 57.4 56.9 56.7 SUM VP1 + VP2 + VP3 100.0 100.0 100.0100.0 100.0 100.0 Purity and summary with storage time = 2 months at −20± 2° C. Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Lane 9Lane 10 Lane 11 Lane 12 Lane 13 Lane 14 Band Band % Band % Band % Band %Band % Band % VP1 16.1 16.5 16.1 16.2 16.2 16.8 VP2 26.6 27.2 26.5 26.726.1 26.6 VP3 57.4 56.3 57.4 57.1 57.7 56.6 SUM VP1 + VP2 + VP3 100.0100.0 100.0 100.0 100.0 100.0 Purity and summary with storage time = 2months at ≤−60° C. Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6Lane 15 Lane 16 Lane 17 Lane 18 Lane 19 Lane 20 Band Band % Band % Band% Band % Band % Band % VP1 16.4 16.5 16.4 16.7 16.9 17.7 VP2 26.5 26.826.4 26.5 26.7 27.3 VP3 57.1 56.6 57.2 56.7 56.5 55.0 SUM VP1 + VP2 +VP3 100.0 100.0 100.0 100.0 100.0 100.0

TABLE 16 Purity and summary with storage time = 3 months at −20 ± 2° C.Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Lane 5 Lane 6 Lane7 Lane 8 Lane 9 Lane 10 Band Band % Band % Band % Band % Band % Band %VP1 19.58 18.95 18.78 18.6 18.69 18.45 VP2 27.6 27.33 27.65 27.32 27.3127.21 VP3 52.82 53.72 53.57 54.08 53.99 54.35 SUM VP1 + VP2 + VP3 100100 100 100 100 100 Purity and summary with storage time = 3 months at≤−60° C. Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Lane 12Lane 13 Lane 14 Lane 15 Lane 16 Lane 17 Band Band % Band % Band % Band %Band % Band % VP1 18.6 18.49 18.91 19.14 19.08 19.07 VP2 27.06 26.627.69 27.04 27.81 27.57 VP3 54.34 54.92 53.4 53.82 53.12 53.36 SUM VP1 +VP2 + VP3 100 100 100 100 100 100

TABLE 17 Purity and summary with storage time = 4 months at +5 ± 3° C.Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Lane 5 Lane 6 Lane7 Lane 8 Lane 9 Lane 10 Band Band % Band % Band % Band % Band % Band %5.39 VP1 15.75 17.96 18.03 18.42 17.99 17.66 VP2 27.73 27.83 28.56 28.2729.06 28.2 VP3 51.12 54.21 53.42 53.31 52.94 54.14 SUM VP1 + VP2 + VP394.6 100 100 100 100 100

TABLE 18 Purity and summary with storage time = 6 months at −20 ± 2° C.Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 lane 5 lane 6 lane7 lane 8 lane 9 lane 10 Band Band % Band % Band % Band % Band % Band %VP1 19.26 19.27 19.01 18.81 18.59 18.72 VP2 27.79 27.64 26.85 27.2526.93 26.83 VP3 52.95 53.09 54.14 53.94 54.48 54.45 SUM VP1 + VP2 + VP3100.0 100.0 100.0 100.0 100.0 100.0 Purity and summary with storage time= 6 months at ≤−60° C. Sample 1 Sample 2 Sample 3 Sample 4 Sample 5Sample 6 lane 12 lane 13 lane 14 lane 15 lane 16 lane 17 Band Band %Band % Band % Band % Band % Band % VP1 18.63 18.33 18.46 18.73 18.818.94 VP2 26.83 26.61 27.29 26.5 26.53 27.16 VP3 54.54 55.07 54.25 54.7754.67 53.9 SUM VP1 + VP2 + VP3 100.0 100.0 100.0 100.0 100.0 100.0

The results of the subvisible particle concentration of material usingMFI are shown in Tables 19-23. This assay was performed to observesubvisible particle generation during storage of AAV material at 5±3° C.for 4 months and at −20±2° C. and ° C. for 6 months.

TABLE 19 Comparison of particles ≥10 μm (number/ml) sample 0 months 4months +5 ± 3° C. 6 months −20 ± 2° C. 6 months ≤−60° C. Sample 1 515.89478.03 606.05 Sample 2 548.45 826.43 748.80 688.69 Sample 3 550.951179.54 1570.22 1450.01 Sample 4 578.50 593.53 668.66 Sample 5 813.915086.30 4299.94 4675.59 Sample 6 1014.25 437.65

TABLE 20 Comparison of particles ≥25 μm (number/ml) sample 0 months 4months +5 ± 3° C. 6 months −20 ± 2° C. 6 months ≤−60° C. Sample 1 100.17117.63 157.77 Sample 2 95.16 127.72 175.30 152.76 Sample 3 107.69 363.13250.43 185.32 Sample 4 120.21 107.69 152.76 Sample 5 127.72 833.94558.47 548.45 Sample 6 272.97 83.00

TABLE 21 Particle concentration at 0 months Measurement ≥1 μm ≥2 μm ≥5μm ≥10 μm ≥25 μm ≥50 μm ≥70 μm Sample 1 29107.85 9408.77 1803.12 515.89100.17 20.03 7.51 Sample 2 34912.89 10653.43 1878.25 548.45 95.16 12.522.50 Sample 3 35030.59 10250.23 1783.08 550.95 107.69 25.04 10.02 Sample4 30908.46 9303.59 1798.11 578.50 120.21 17.53 5.01 Sample 5 48681.7012366.39 2376.61 813.91 127.72 20.03 2.50 Sample 6 39713.69 11710.252784.82 1014.25 272.97 67.62 35.06

TABLE 22 Particle concentration after 4 months at +5 ± 3° C. Measurement≥1 μm ≥2 μm ≥5 μm ≥10 μm ≥25 μm ≥50 μm ≥70 μm Sample 2 +5 ± 3° C.22999.78 11469.84 3551.14 826.43 127.72 22.54 2.50 Sample 3 +5 ± 3° C.11504.90 5088.80 2033.52 1179.54 363.13 72.63 40.07 Sample 4 +5 ± 3° C.28096.10 9498.93 1808.13 593.53 107.69 27.55 10.02 Sample 5 +5 ± 3° C.19516.26 11943.16 7798.49 5086.30 833.94 75.13 22.54 Sample 6 +5 ± 3° C.20710.38 6235.25 1466.38 437.65 83.00 22.64 7.55

TABLE 23 Particle concentration after 6 months at −20 ± 2° C. and ≤−60°C. Measurement ≥1 μm ≥2 μm ≥5 μm ≥10 μm ≥25 μm ≥50 μm ≥70 μm Sample 1−20° C. 24131.83 6790.05 1501.67 478.03 117.63 45.05 12.51 Sample 1≤−60° C. 24144.26 6836.83 1510.11 606.05 157.77 35.06 22.54 Sample 2−20° C. 44356.72 16899.23 3591.21 748.80 175.30 42.57 12.52 Sample 2≤−60° C. 32438.61 9433.82 2036.02 688.69 152.76 40.07 22.54 Sample 3−20° C. 51606.76 9498.93 3032.75 1570.22 250.43 75.13 55.10 Sample 3≤−60° C. 43041.95 9493.92 2874.97 1450.01 185.32 35.06 17.53 Sample 4≤−60° C. 31191.45 8807.73 1930.84 668.66 152.76 32.56 10.02 Sample 5−20° C. 82750.63 17525.31 7695.81 4299.94 558.47 50.09 25.04 Sample 5≤−60° C. 77216.06 19578.87 8812.74 4675.59 548.45 50.09 22.54

Results of osmolality measurements are shown in Table 24.

TABLE 24 Osmolality measurements Name and Formulation mOsmol/kg Buffer1⁷: Chatham buffer + 0.005% PS80 990 Buffer 2⁷: 20 mM His, 70 mM NaCl,5% Sucrose, 0.005% 309 PS80 Buffer 3⁷: 20 mM His, 60 mM NaCl, 110 mMMannit, 296 35 mM Trehalose Dihydrate, 0.005% PS80 Buffer 4⁷: 10 mM His,100 mM NaCl, 50 mM Glycine, 5% 386 Trehalose Dihydrate, 0.005% PS80Buffer 5⁷: 15 mM Na3Cit buffer, 15 mM Glycine, 20 g 212 Mannit, 10 gTrehalose Dihydrat, 0.005% PS80 Buffer 6⁷: 80 mM TRIS, Dextran T10 andT40, 6.9% 417 Sucrose. 0.005% PS80

In conclusion, there were no significant differences among the sixformulations for AAV8 titration ELISA after 4 months at 5±3° C. andafter 6 months at −20±2° C. and ≤−60° C. Appearance complies forFormulations 2-5, while a few particles formed for Formulation 1 and aslight yellow color was observed for Formulation 6. All formulations,except Formulation 6 and the control, showed stability during storagetime for all temperatures as tested by SDS-PAGE (densiometric method).Based on appearance, sub-visible particle assay (MFI) data, andosmolality values, Formulations 2, 3, and 4 were selected for furtherdevelopment.

Example 2

This example demonstrates the additional testing and further developmentof three formulations.

Formulations 2, 3, and 4 (Buffers 2, 3, and 4, Samples 2, 3, and 4) weremanufactured as essentially described in Example 1. Briefly, a singlelot was diluted with PBS/NaCl/Sorbitol buffer to general 90 ml. Thesolution was spiked with 0.005% Croda super refined PS80 and aliquotedinto 3 parts. The buffer was changed to one of the three differentbuffers or control buffer via Slide-A-Lyzer dialysis cassettes and thisstep was repeated for a total of 3 times with 100 fold volume of thesample. The 3 different solutions were filled into 2 mL Schott Type Iglass vials with a SiO₂ layer and stored at the temperatures describedbelow.

In this example, “Buffer 1” refers to Formulation/Buffer/Sample 2 ofExample 1, “Buffer 2” refers to Formulation/Buffer/Sample 4 of Example1, “Buffer 3” refers to Formulation/Buffer/Sample 3 of Example 1, and“Buffer 4” refers to a PBS/NaCl/Sorbitol buffer control. For clarity,Tables 25-28 describe Buffers 1-3 and the PBS/NaCl/Sorbitol buffercontrol of this example.

TABLE 25 Buffer 1 (Formulation 2) Concentration g/kg ExcipientConcentration [mM] buffer L-histidine 20 3.103 NaCl 70 4.09 Sucrose   5% 50.00 Croda super refined Tween 80 0.005% 0.05 (PS80) Purifiedwater add to 1 kg pH value 7.5

TABLE 26 Buffer 2 (Formulation 4) Concentration g/kg ExcipientConcentration [mM] buffer L-histidine 10 1.552 NaCl 100  5.844 Glycine50 3.754 Trehalose Dihydrate    5% 50 Croda super refined Tween 800.005% 0.05 Purified water add to 1 kg pH value 7.0 ± 0.1

TABLE 27 Buffer 3 (Formulation 3) Concentration g/kg ExcipientConcentration [mM] buffer L-histidine 20 3.103 NaCl 60 3.506 Mannitol110 20.039 Trehalose Dihydrate 35 13.24 Croda super refined Tween 800.005% 0.05 (PS80) Purified water add to 1 kg pH value 7.0 ± 0.1

TABLE 28 PBS/NaCl/Sorbitol Buffer Control Concentration g/kg ExcipientConcentration [mM] buffer Sodium chloride 350    20.45 Potassiumchloride 2.68 0.2 Disodiumhydrogenphosphate- 8.09 1.44 DihydrateMonopotassium phosphate 1.47 0.2 D-Sorbitol    5% 50.00 Croda superrefined Tween 80 0.005% 0.05 (PS80) 25% HCl pH adjustment 1M NaOHPurified water add to 1 kg pH value 7.4 ± 0.3

Medium- to long-term stability studies at ° C. (set point: −80° C.) andat −20±2° C. for up to 5 months (0, 1, 2, 3, and 5 months) and at 5±3°C. for up to 4 months (0, 1, 2, and 4 months) were performed. Theformulations were stored in 2 ml Schott Type I glass vials with a SiO₂layer. Each vial was filled with 0.3 mL of a formulation. The analyticaltests listed in Table 29 were performed and the test time points foreach assay at the indicated storage temperatures are shown in Tables 30and 31. A “-” in Table 30 or 31 means no samples were taken.

TABLE 29 Analytical assays Detection by analytical method/ QualityAttribute principle General Polysorbate 80 Appearance pH values ActivityIn vitro Biopotency In vivo Biopotency FIX-qPCR Total particle titer(empty + full) Total rAAV8 particle ELISA AAV purity SDS-PAGE(fluorescence staining, Chemical degradation 4-12% BisTris gel withFlamingo Stain) Physical degradation WAX [Weak Anion Exchange HPLC](damaged capsids or fragments) SEC [Size exclusion HPLC]

TABLE 30 Study outline with storage at −20 ± 2° C. and ≤−60° C. (setpoint: −80° C.) Test Time Total Sample Point [months] Number of TestVolume 0 1 2 3 5 Samples SDS-PAGE (fluorescence 0.3 ml x x x x x 36staining, 4-12% BisTris gel with Flamingo Stain) WAX 0.3 ml x x x x x 36[Weak Anion Exchange HPLC] SEC [Size exclusion HPLC] 0.3 ml x x x x x 36In vitro Biopotency 0.3 ml x x x x x 36 In vivo Biopotency 0.3 ml x x —— x 20 FIX-qPCR 0.3 ml x x x x x 36 Total rAAV8 particle 2 × x x x x x72 ELISA 0.3 ml Appearance 1 ml x x — — — 136 pH value x x — — — 12Polysorbate 80 0.3 ml — — — — x 3

TABLE 31 Study outline with storage at +5 ± 3° C. Total Number SampleTest Time Point [months] of Test Volume 0 1 2 4 Samples SDS-PAGE(fluorescence staining, 4-12% 0.3 ml x x x x 16 BisTris gel withFlamingo Stain) WAX 0.3 ml x x x x 16 [Weak Anion Exchange HPLC] SEC[Size exclusion HPLC] 0.3 ml x x x x 16 In vitro Biopoteney 0.3 ml x x xx 16 In vivo Biopotency 0.3 ml x x — x 12 FIX-qPCR 0.3 ml x x x x 16Total rAAV8 particle ELISA 2 × x x x x 32 0.3 ml Appearance   1 ml x x xx 56 pH value x x — — 8

Results of the ELISA are shown in Table 32. AAV8 titration ELISA showeda massive amount of material loss for Buffer 4 (control) during storageat 5±3° C. Compare 5.61×10¹² [cp/ml] at 0 months to 2.71×10¹² after 4months. No additional losses were observed for the same material storedat −20±2° C. and ≤−60° C. Buffers and 1 and 2 did not exhibit anymaterial loss. Buffer 3 showed a lower starting material amount thatdoes not decrease during storage time.

TABLE 32 AAV8 titration ELISA AAV8 Titration 1 month 2 months 4 monthsELISA [cp/ml] at +5 ± 3° C. 0 months at +5 ± 3° C. at +5 ± 3° C. at +5 ±3° C. BUFFER 1 9.39E+12 7.46E+12 9.33E+12 8.56E+12 BUFFER 2 1.01E+137.46E+12 9.17E+12 9.90E+12 BUFFER 3 8.05E+12 6.69E+12 7.55E+12 8.03E+12BUFFER 4 5.61E+12 4.80E+12 3.82E+12 2.71E+12 1 month 2 months 3 months 5months AAV8 Titration at at at at ELISA [cp/ml] at −20 ± 2° C. 0 months−20 ± 2° C. −20 ± 2° C. −20 ± 2° C. −20 ± 2° C. BUFFER 1 9.39E+127.59E+12 8.73E+12 8.67E+12 9.83E+12 AAV8 Titration 1 month 2 months 4months ELISA [cp/ml] at +5 ± 3° C. 0 months at +5 ± 3° C. at +5 ± 3° C.at +5 ± 3° C. BUFFER 2 1.01E+13 7.81E+12 8.78E+12 9.17E+12 1.00E+13BUFFER 3 8.05E+12 6.60E+12 7.55E+12 7.52E+12 8.17E+12 BUFFER 4 5.61E+125.35E+12 5.68E+12 5.17E+12 6.79E+12 AAV8 Titration 1 month 2 months 3months 5 months ELISA [cp/ml] at ≤−60° C. 0 months at ≤−60° C. at ≤−60°C. at ≤−60° C. at ≤−60° C. BUFFER 1 9.39E+12 7.35E+12 9.13E+12 8.58E+129.62E+12 BUFFER 2 1.01E+13 7.35E+12 9.46E+12 8.82E+12 9.98E+12 BUFFER 38.05E+12 6.14E+12 7.77E+12 7.22E+12 8.59E+12 BUFFER 4 5.61E+12 4.91E+126.45E+12 5.26E+12 6.00E+12

The results of the qPCR are shown in Table 33. All data generated withthis assay confirm the ELISA and in vitro biopotency assay. Buffer 4shows a high amount of test variation.

TABLE 33 FIX-qPCR [vg/ml] FIX-qPCR [vg/ml] at +5 ± 3° C. 1 month 2months 4 months 0 months at +5 ± 3° C. at +5 ± 3° C. at +5 ± 3° C.BUFFER 1 6.11E+12 4.34E+12 1.74E+12 4.65E+12 BUFFER 2 4.34E+12 4.60E+125.16E+12 4.14E+12 BUFFER 3 3.66E+12 3.53E+12 3.86E+12 3.49E+12 BUFFER 41.62E+12 1.63E+12 1.92E+12 4.98E+12 FIX-qPCR [vg/ml] at −20 ± 2° C. 1month 2 months 3 months 5 months 0 months at −20 ± 2° C. at −20 ± 2° C.at −20 ± 2° C. at −20 ± 2° C. BUFFER 1 6.11E+12 3.42E+12 4.42E+124.10E+12 4.07E+12 BUFFER 2 4.34E+12 3.57E+12 4.12E+12 4.01E+12 4.20E+12BUFFER 3 3.66E+12 2.23E+12 3.44E+12 3.51E+12 3.79E+12 BUFFER 4 1.62E+124.33E+12 2.70E+12 3.29E+12 2.06E+12 FIX-qPCR [vg/ml] at ≤−60° C. 1 month2 months 3 months 5 months 0 months at ≤−60° C. at ≤−60° C. at ≤−60° C.at ≤−60° C. BUFFER 1 6.11E+12 4.59E+12 4.67E+12 4.06E+12 4.35E+12 BUFFER2 4.34E+12 3.78E+12 4.77E+12 2.78E+12 4.37E+12 BUFFER 3 3.66E+122.57E+12 3.68E+12 3.48E+12 3.37E+12 BUFFER 4 1.62E+12 1.47E+12 1.97E+121.60E+12 1.86E+12

The results of the % aggregate measurements are shown in Table 34.Decreasing aggregate values were seen after storage for 4 months at 5±3°C. Buffer 4 showed an increase from 1.55% to 3.39% after 3 months and noaggregates after 4 months. Buffer 3 reached a high of 6.64% after 2months at −60° C. that dropped to 3.25% after 3 months and 3.94% after 5months.

TABLE 34 Aggregates [%] Aggregates [%] at +5 ± 3° C. 1 month 2 months 4months 0 months at +5 ± 3° C. at +5 ± 3° C. at +5 ± 3° C. BUFFER 1 2.392.34 2.26 1.40 BUFFER 2 2.46 2.13 2.55 1.36 BUFFER 3 2.65 2.49 2.79 1.39BUFFER 4 1.55 3.39 3.39 0.00 Aggregates [%] at −20 ± 2° C. 1 month 2months 3 months 5 months 0 months at −20 ± 2° C. at −20 ± 2° C. at −20 ±2° C. at −20 ± 2° C. BUFFER 1 2.39 2.35 2.49 2.46 2.81 BUFFER 2 2.462.30 2.48 2.42 2.47 BUFFER 3 2.65 2.62 2.91 2.79 2.85 BUFFER 4 1.55 3.063.24 3.73 3.66 Aggregates [%] at ≤−60° C. 1 month 2 months 3 months 5months 0 months at ≤−60° C. at ≤−60° C. at ≤−60° C. at ≤−60° C. BUFFER 12.39 2.24 2.26 2.12 2.56 BUFFER 2 2.46 2.20 2.50 2.57 2.39 BUFFER 3 2.653.39 6.64 3.25 3.94 BUFFER 4 1.55 2.28 2.01 2.90

The results of the weak anion exchange (WAX) assay which measures fullcapsids did not exhibit any meaningful differences for all testing timepoints and storage temperatures (Table 35).

TABLE 35 Full Capsids [%] Full Capsids [%] at +5 ± 3° C. 1 month 2months 4 months 0 months at +5 ± 3° C. at +5 ± 3° C. at +5 ± 3° C.BUFFER 1 61 62 67 62 BUFFER 2 62 64 65 61 BUFFER 3 63 62 64 63 BUFFER 462 62 66 58 Full Capsids [%] at −20 ± 2° C. 1 month 2 months 3 months 5months 0 months at −20 ± 2° C. at −20 ± 2° C. at −20 ± 2° C. at −20 ± 2°C. BUFFER 1 61 64 68 63 62 BUFFER 2 62 63 66 61 61 BUFFER 3 63 62 66 6361 BUFFER 4 62 62 64 63 60 Full Capsids [%] at ≤−60° C. 1 month 2 months3 months 5 months 0 months at ≤−60° C. at ≤−60° C. at ≤−60° C. at ≤−60°C. BUFFER 1 61 64 64 61 60 BUFFER 2 62 62 65 63 63 BUFFER 3 63 63 65 6261 BUFFER 4 62 63 66 62 61

The results of the in vitro biopotency assay are shown in Table 36.Similar fluctuating values are shown for Buffer 1, Buffer 2, and Buffer3 in the range of test variation. Buffer 4 shows a high decrease afterstorage at 5±3° C. for 4 months. Comparable results were detected forall other buffers, testing time points and temperatures.

TABLE 36 In vitro Biopotency [BPU] In vitro Biopotency [BPU] at +5 ± 3°C. 1 month 2 months 4 months 0 months at +5 ± 3° C. at +5 ± 3° C. at +5± 3° C. BUFFER 1 1.05 0.87 1.05 1.22 BUFFER 2 0.85 1.02 1.02 1.07 BUFFER3 0.78 0.90 1.14 1.20 BUFFER 4 0.67 0.65 0.57 0.13 In vitro Biopotency[BPU] at −20 ± 2° C. 1 month 2 months 3 months 5 months 0 months at −20± 2° C. at −20 ± 2° C. at −20 ± 2° C. at −20 ± 2° C. BUFFER 1 1.05 0.831.23 1.09 1.24 BUFFER 2 0.85 0.84 1.11 0.97 1.35 BUFFFR 3 0.78 0.68 0.941.13 1.25 BUFFER 4 0.67 1.93 1.76 0.79 1.18 In vitro Biopotency [BPU] at≤−60° C. 1 month 2 months 3 months 5 months 0 months at ≤−60° C. at≤−60° C. at ≤−60° C. at ≤−60° C. BUFFER 1 1.05 1.15 1.10 1.01 1.34BUFFER 2 0.85 1.09 0.80 0.96 1.21 BUFFER 3 0.78 0.83 0.96 0.94 1.12BUFFER 4 0.67 0.80 0.98 0.68 1.08

The results of the in vivo biopotency assay are shown in Table 37.Results for Buffers 1-3 show fluctuating results in the range of testvariation. Buffer 4 decreases from 3.70 [11.1/m1] at 0 months to 1.33[11.1/m1] after 4 months at 5±3° C. and increases from 3.79 [11.1/m1] at0 months to 6.73 [11.1/m1] after 4 months at ≤−60° C. Day 7 results weresimilar to Day 14 results. The results after 1 month storage at ≤−60° C.are taken as a reference value representing the 0 month for all storagetemperatures.

TABLE 37 In vivo Biopotency [Iu/ml] day 14 results 1 month at 2 monthsat 3 months at 4 months at In vivo Biopotency 0 months +5 ± 3° C., +5 ±3° C., +5 ± 3° C., +5 ± 3° C., [IU/ml] at +5 ± 3° C. day 14 day 14 day14 day 14 day 14 BUFFER 1 3.48 ± 1.28 5.91 ± 1.33 4.82 ± 0.63 4.76 ±0.55 BUFFER 2 4.99 ± 0.74 5.49 ± 0.64 5.27 ± 0.84 5.36 ± 1.80 BUFFER 34.43 ± 1.15 6.42 ± 0.89 4.64 ± 1.42 5.58 ± 0.68 BUFFER 4 3.79 ± 0.703.43 ± 0.61 2.77 ± 0.44 1.33 ± 0.26 1 month at 2 months at 3 months at 4months at 5 months at In vivo Biopotency 0 months −20 ± 2° C., −20 ± 2°C., −20 ± 2° C., −20 ± 2° C., −20 ± 2° C., [IU/ml] at −20 ± 2° C. day 14day 14 day 14 day 14 day 14 day 14 BUFFER 1 3.48 ± 1.28 5.09 ± 2.25 4.97± 1.30 5.28 ± 0.55 BUFFER 2 4.99 ± 0.74 5.85 ± 0.74 4.88 ± 1.44 5.04 ±0.70 BUFFER 3 4.43 ± 1.15 4.34 ± 1.62 5.16 ± 0.79 5.03 ± 1.00 BUFFER 43.79 ± 0.70 3.42 ± 1.46 4.20 ± 1.74 4.39 ± 0.76 1 month at 2 months at 3months at 4 months at 5 months at In vivo Biopotency 0 months ≤−60° C.,≤−60° C., ≤−60° C., ≤−60° C., ≤−60° C., [IU/ml] at ≤−60° C. day 14 day14 day 14 day 14 day 14 day 14 BUFFER 1 3.48 ± 1.28 3.48 ± 1.28 5.99 ±1.58 BUFFER 2 4.99 ± 0.74 4.99 ± 0.74 5.68 ± 1.02 BUFFER 3 4.43 ± 1.154.43 ± 1.15 4.63 ± 1.33 BUFFER 4 3.79 ± 0.70 3.79 ± 0.70 6.73 ± 0.90

TABLE 38 In vivo Biopotency [IU/ml] day 7 results 1 month at 2 months at3 months at 4 months at In vivo Biopotency 0 months ±5 ± 3° C., ±5 ± 3°C., ±5 ± 3° C., ±5 ± 3° C., [IU/ml] at ±5 ± 3° C. day 7 day 7 day 7 day7 day 7 BUFFER 1 2.35 ± 0.79 3.86 ± 0.93 3.15 ± 0.29 2.70 ± 0.32 BUFFER2 3.41 ± 0.55 3.31 ± 0.68 3.81 ± 0.74 3.46 ± 0.23 BUFFER 3 3.29 ± 0.253.58 ± 0.59 3.09 ± 0.76 3.12 ± 0.36 BUFFER 4 2.30 ± 0.48 2.00 ± 0.401.68 ± 0.30 0.80 ± 0.04 1 month at 2 months at 3 months at 4 months at 5months at In vivo Biopotency 0 months −20 ± 2° C., −20 ± 2° C., −20 ± 2°C., −20 ± 2° C., −20 ± 2° C., [IU/ml] at −20 ± 2° C. day 7 day 7 day 7day 7 day 7 day 7 BUFFER 1 2.35 ± 0.79 3.20 ± 1.40 2.91 ± 0.81 3.29 ±0.56 BUFFER 2 3.41 ± 0.55 3.60 ± 0.55 3.01 ± 0.80 3.01 ± 0.64 BUFFER 33.29 ± 0.25 3.04 ± 1.11 3.12 ± 0.56 3.18 ± 0.69 BUFFER 4 2.30 ± 0.481.84 ± 0.80 0.80 ± 0.04 2.46 ± 0.45 1 month at 2 months at 3 months at 4months at 5 months at In vivo Biopotency 0 months day ≤−60° C., ≤−60°C., ≤−60° C., ≤−60° C., ≤−60° C., [IU/ml] at ≤−60° C. day 7 day 7 day 7day 7 day 7 day 7 BUFFER 1 2.35 ± 0.79 2.35 ± 0.79 4.02 ± 1.11 BUFFER 23.41 ± 0.55 3.41 ± 0.55 3.58 ± 1.07 BUFFER 3 3.29 ± 0.25 3.29 ± 0.972.67 ± 0.74 BUFFER 4 2.30 ± 0.48 2.30 ± 0.48 3.53 ± 0.46

The results of the SDS PAGE densiotometric assay are shown in Tables39-44. No additional bands were observed during storage at 5±3° C. forup to 4 months and for storage at −20±2° C. and ≤−60° C. for up to 5months.

TABLE 39 Purity and summary at 0 months storage HT6AR00G (Ref) Sample 1Sample 2 Sample 3 Sample 4 10 μg/ml Mean Value Mean Value Mean ValueMean Value Lane 3 L5-L7 L9-L11 L13-L15 L17-L19 Vector protein Band %Band % Band % Band % Band % VP1 11.4 17.5 17.5 17.2 15.6 VP2 10.9 19.920.9 20.4 18.8 VP3 77.7 62.6 61.6 62.4 65.6 SUM VP1 + VP2 + VP3 100 100100 100 100 (Purity)

TABLE 40 Purity and summary at 1 month storage Reference +5 ± 3° C. 1month −20 ± 2° C. 1 month HT6AR00G Buffer Buffer Buffer Buffer BufferBuffer 10 μg/ml 1 2 3 4 1 2 Lane Lane Lane Lane Lane Lane Lane 3 5 6 7 810 11 Vector Band Band Band Band Band Band Band protein % % % % % % %VP1 7.49 15.63 15.95 15.82 14.06 14.91 15.34 VP2 10.96 18.16 18.18 18.5616.12 17.05 18.33 VP3 81.54 66.21 65.88 65.62 69.82 68.04 66.32 SUM 100100 100 100 100 100 100 VP1 + VP2 + VP3 (Purity) −20 ± 2° C. 1 month −60± 2° C. 1 month Buffer Buffer Buffer Buffer Buffer Buffer 3 4 1 2 3 4Lane Lane Lane Lane Lane Lane 12 13 15 16 17 18 Vector Band Band BandBand Band Band protein % % % % % % VP1 15.72 14.22 15.31 15.27 14.9914.35 VP2 18.14 17.65 19.18 19.48 18.33 17.36 VP3 66.14 68.13 65.5165.25 66.69 68.29 SUM 100 100 100 100 100 100 VP1 + VP2 + VP3 (Purity)

TABLE 41 Purity and summary at 2 months storage Reference +5 ± 3° C. 2month −20 ± 2° C. 2 month −60 ± 2° C. 2 month HT6AR00G Buffer BufferBuffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer 10μg/ml 1 2 3 4 1 2 3 4 1 2 3 4 Lane Lane Lane Lane Lane Lane Lane LaneLane Lane Lane Lane Lane 3 5 6 7 8 10 11 12 13 15 16 17 18 Vector BandBand Band Band Band Band Band Band Band Band Band Band Band protein % %% % % % % % % % % % % VP1 6.33 12.14 12.92 12.1 8.7 12.28 14.09 11.8 10.82 12.28 12.61 12.25 10.96 VP2 7.46 19.79 19.24 17.78 14.62 18.8418.26 16.97 15.28 18.77 19.45 17.87 16.08 VP3 86.21 68.07 67.84 70.1276.68 68.87 67.65 71.24 73.9 68.95 67.95 69.88 72.96 SUM 100 100   100    100 100 100    100 100    100 100    100 100    100    VP1 +VP2 + VP3 (Purity)

TABLE 42 Purity and summary at 3 months storage Reference HT6AR00G −20 ±2° C. 3 month −60 ± 2° C. 3 month 10 μg/ml Buffer 1 Buffer 2 Buffer 3Buffer 4 Buffer 1 Buffer 2 Buffer 3 Buffer 4 Lane 3 Lane 5 Lane 6 Lane 7Lane 8 Lane 10 Lane 11 Lane 12 Lane 13 Band Band Band Band Band BandBand Band Band Vector protein % % % % % % % % % VP1 7.1 16.73 16.7516.63 14.98 15.93 16 16.05 13.3 VP2 6.56 20.94 21.9 21.02 17.77 21.0721.89 19.95 16.14 VP3 86.34 62.32 61.35 62.36 67.25 63 62.11 64.01 70.55SUM 100 100 100 100 100 100 100 100 100 VP1 + VP2 + VP3 (Purity)

TABLE 43 Purity and summary at 3 months storage Reference HT6AR00G +5 ±3° C. 4 month 10 μg/ml Buffer 1 Buffer 2 Buffer 3 Buffer 4 Lane 3 Lane 5Lane 6 Lane 7 Lane 8 Vector protein Band % Band % Band % Band % Band %VP1 12.13 16.16 17.45 16.44 11.35 VP2 14.66 24.2 22.36 21.25 10.1 VP373.2 59.65 60.19 62.3 78.55 SUM VP1 + VP2 + 100 100 100 100 100 VP3(Purity)

TABLE 44 Purity and summary at 5 months storage Reference HT6AR00G −20 ±2° C. 5 month −60 ± 2° C. 5 month 10 μg/ml Buffer 1 Buffer 2 Buffer 3Buffer 4 Buffer 1 Buffer 2 Buffer 3 Buffer 4 Lane 3 Lane 5 Lane 6 Lane 7Lane 8 Lane 10 Lane 11 Lane 12 Lane 13 Band % Band % Band % Band % Band% Band % Band % Band % Band % VP1 11.51 17.45 17.52 17.2 14.68 16.6516.91 16.83 15.24 VP2 16.44 22.43 23.27 22.5 19.7 22.46 22.69 22.3820.38 VP3 72.05 60.12 59.2 60.3 65.62 60.89 60.4 60.79 64.37 SUM 100 100100 100 100 100 100 100 100 VP1 + VP2 + VP3 (Purity)

Results of the appearance test are shown in Table 45-48, wherein theclassification of particles inspection with an unaided eye were asfollows: Level A=no particles visible; Level B=small single particles,barely visible; Level C=small single particles, easily visible; Level Dmany small particles, easily visible; and Level E=particles visiblegreater than or equal to 1 mm. The appearance of all samples after 1month at 5±3° C., −20±2° C., and ≤−60° C. (set point: −80° C.) exhibitedclear, colorless solutions without visible particles (denoted below andthroughout the specification as “complies”).

TABLE 45 Appearance during 48 hours at +25 ± 2° C. Appearance starting1.5 3 4.5 6 24 48 at +25 ± 2° C. material hours hours hours hours hourshours Concentration of AAV8 particles: 8.8E+12 [cp/ml] BUFFER 1 A A A AA A A BUFFER 2 A A A A A A A BUFFER 3 A A A A A A A BUFFER 4 A A A A-BA-B A A-B Concentration of AAV8 particles: 17.6E+12 [cp/ml] BUFFER 1 A AA A A A A BUFFER 2 A A A A A A A BUFFER 3 A A A A A A A BUFFER 4 A A BA-B A-B A-B A-B

TABLE 46 Appearance during 4 months at +5 ± 3° C. starting materialshort time starting material after sterile after 6 days at 1 2 4 4Appearance at +5 ± 3° C. filtration +5 ± 3° C. month months monthsmonths Concentration of AAV8 particles: 8.8E+12 [cp/ml] BUFFER 1 Acomplies to A A A complies BUFFER 2 A criteria A A A complies BUFFER 3 AA A A complies BUFFER 4 A clear, colorless, A-B A-B A-B complies withvisible particles (2 fibers) (2 fibers) Concentration of AAV8 particles:17.6E+12 [cp/ml] BUFFER 1 A complies to A A A complies BUFFER 2 Acriteria A A A complies BUFFER 3 A A A A complies BUFFER 4 A clear,colorless, A-B A-B A-B doesn't with visible particles (4 fibers) (4fibers, comply, >5 white particles) particles > 10

TABLE 47 Appearance during 6 months at −20 ± 2° C. starting startingmaterial material short after time freezing after at starting materialAppearance at sterile −20 ± after freezing 1 2 3 4 6 6 −20 ± 2° C.filtration 2° C. at −20 ± 2° C. month months months months months monthsConcentration of AAV8 particles: 8.8E+12 [cp/ml] BUFFER 1 A A complies AA A complies A complies BUFFER 2 A A-B complies A A A complies Acomplies BUFFER 3 A A complies A A A-B complies A-B complies BUFFER 4 AB (1 clear, colorless, A-B A-B B doesn't C clear, particle) with visibleparticles (3 fibers) (>5 comply, (>6 colorless particles) white fibers)solution particles > with 5 visible particles > 10 Concentration of AAV8particles: 17.6E+12 [cp/ml] BUFFER 1 A A-B complies A A A-B complies A-Bcomplies BUFFER 2 A A-B complies A A A complies A complies BUFFER 3 AA-B complies A A A-B complies A-B complies BUFFER 4 A B clear,colorless, A-B A-B C (3 doesn't C (3 clear, with visible particles (2fibers) fibers, comply, fibers, colorless >5 white >10 solutionparticles) particles > particles) with 5 visible particles > 10

TABLE 48 Appearance during 6 months at 6° C. starting material startingshort time material after after freezing sterile at − ≤ 1 2 3 6 6Appearance at ≤−60° C. filtration −60° C. month months months monthsmonths Concentration of AAV8 particles: 8.8E+12 [cp/ml] BUFFER 1 A A A AA A complies BUFFER 2 A A A A A A complies BUFFER 3 A A A A A A compliesBUFFER 4 A A-B A-B A-B A-B A-B complies (3 fibers) (4 fibers)Concentration of AAV8 particles: 17.6E+12 [cp/ml] BUFFER 1 A A A A A Acomplies BUFFER 2 A A A A A A complies BUFFER 3 A A A A A A compliesBUFFER 4 A A-B A-B A-B A-B B-C clear, (3 fibers) (4 fibers) (>5colorless fibers) solution with visible particles > 10

pH values for the buffers/formulations at 0 months and after 1 monthstorage at the indicated temperatures are shown in Table 49.

TABLE 49 pH values at 1 month pH values at +5 ± 3° C. 1 month target pHvalue 0 months at +5 ± 3° C. BUFFER 1 7.5 7.45 7.46 BUFFER 2 7.0 6.956.94 BUFFER 3 7.0 7.00 6.98 BUFFER 4 7.4 7.34 7.31 pH values at −20 ± 2°C. 1 month target pH value 0 months at −20 ± 2° C. BUFFER 1 7.5 7.457.44 BUFFER 2 7.0 6.95 6.96 BUFFER 3 7.0 7.00 6.99 BUFFER 4 7.4 7.347.33 pH values at ≤−60° C. 1 month target pH value 0 months at ≤−60° C.BUFFER 1 7.5 7.45 7.43 BUFFER 2 7.0 6.95 6.95 BUFFER 3 7.0 7.00 6.98BUFFER 4 7.4 7.34 7.33

Polysorbate 80 (PS80) measurements were performed after storage for 5months at ≤−60° C. The results (shown in Table 50) confirm the amount ofPS80 was not reduced after storage for 5 months at ≤−60° C.

TABLE 50 Polysorbate 80 (HPLC method) recovery Samples μg/ml [%] targetconcentration 50 100% sample 1 (Buffer 1 - Formulation 2) 62.6 125%sample 2 (Buffer 2 - Formulation 4) 65.7 131% sample 3 (Buffer 3 -Formulation 3) 47.8 96%

Based on the data in this study, no significant different was seen inthe stability profile of AAV8 when formulated in any one of the threebuffers/formulations tested. Therefore, all three may be consideredsuitable for the storage of AAV8 gene therapy products.

Example 3

This example demonstrates an additional investigation of twoformulations for their adsorption to the inner surfaces of differentvials and tubes.

Buffers 1 (Formulation 2) and 2 (Formulation 4) described in Example 2were tested for adsorption to the inner surfaces of different vials andtubes. Buffer 1 (1717.37×10¹¹ cp/ml) and Buffer 2 (1431.94×10¹¹ cp/ml)were diluted to a target concentration of 1.1×10¹³ cp/ml and sterilefiltered with a PALL EKV filter. The samples were loaded into 2 ml glassvials and frozen, for future reference. The materials were then filledinto vials and tubes as described in Table 51.

TABLE 51 Sample container Filing volume   2 ml SiO₂ glass vial itemnumber 3000375 0.3 ml  10 ml SiO₂ glass vial item number 3000727 5.6 ml1.8 ml Nunc ® Cryo Tubes (PP) 0.5 ml 0.5 ml Eppendorf safe lock tubes ®(PP) 0.2 ml 1.5 ml Eppendorf safe lock tubes ® (PP) 0.5 ml 0.5 mlEppendorf Protein LoBind tubes ® (PP) 0.2 ml 1.5 ml Eppendorf ProteinLoBind tubes ® (PP) 0.5 ml

The samples were stored at 25±2° C. for 8 hours and then tested viaPDTS/AAV8 titration ELISA. The results of the assay are shown in Table52.

TABLE 52 Buffer 1 Recovery Buffer2 Recovery storage container [cp/ml][%] [cp/ml] [%]   2 ml SiO₂ vial, starting 1.61E+13 100 1.04E+13 100mateial after 8 hours at +25 ± 2° C.   2 ml SiO₂ vial 1.53E+13 951.05E+13 101  10 ml SiO₂ vial 1.61E+13 100 1.03E+13 99 1.8 ml Nunc1.56E+13 97 1.03E+13 99 0.5 ml safe lock tube 1.60E+13 99 1.01E+13 971.5 ml safe lock tube 1.69E+13 105 1.06E+13 102 0.5 ml LowBind tube1.57E+13 97 9.85E+12 95 1.5 ml LowBind tube 1.62E+13 100 1.05E+13 101

These results demonstrate that greater than 90% of the starting genetherapy material is recoverable from the 2 ml SiO₂ vials. No adsorptionwas seen, and no significant difference was seen among the two testbuffers.

Example 4

This example demonstrates the stability of various AAV subtypes inFormulation 4.

AAV subtypes AAV 2, AAV 5, AAV 8, and AAV 9 were produced (University ofMassachusetts at Worcester Medical School Vector Core Facility) andstored in Formulation 4 (as described in Examples 1-3), then tested forappearance to confirm no visible particles are generated and by ITR-qPCRto quantify vector genome for dosing in in vitro biopotency assay. TheseAAV subtypes each comprised an eGFP package insert that encoded GreenFluorescence Protein (CB6-Pl-eGFP). Testing was conducted at time point0 and again at 6 weeks after storage at +5±3° C.

Two milliliter samples provided in a PBS based buffer (1× PBS+0.001%Pluoronic F+68) with concentration of >5E+12 vg/ml AAV particles werethawed at ambient temperature and diluted to 4 ml(approximately >2.5E+12 vg/ml) with Formulation 4. The approximately0.0025% Polysorbate 80 from Formulation 4 prevented excessive materialloss via adsorption (e.g., on the inner surfaces of the equipment).Total buffer of each of the samples were changed to Formulation 4 usingSlide-A-Lyzer® 10K dialysis cassettes, and this step was performed threetimes with 100-fold volume, with dialysis taking place for a minimum of4 hours for each step. After the buffer was changed to Formulation 4,the samples were taken for the initial 0 weeks testing time point andthen storage at +5±3° C. for 6 weeks.

There were no visible particles observed for the different AAV subtypesstored in Formulation 4, even after storage for 6 weeks (Table 53).Thus, it is possible that the excipients observed at time 0 wereresolved by 6 weeks.

TABLE 53 Appearance during storage in Formulation 4 at +5 ± 3° C.Testing Time Points Subtype 0 weeks 6 weeks AAV2 Colorless solutionwithout particles, cloudy AAV5 Colorless solution without Clearcolorless solution without particles, very slightly cloudy particlesAAV8 Colorless solution without Clear colorless solution withoutparticles, slightly cloudy particles AAV9 Clear colorless solution Clearcolorless solution without without particles particles

While the recovery of AAV2 was low, which could be due to thevariability of the ITR-qPCR assay (ITR-qPCR assay has test variations of±0.5 log), the other AAV subtypes are stable for the 6 weeks tested. Useof Formulation 4 resulted in surprising stability of the AAV8 subtype.

TABLE 54 ITR-qPCR [vg/ml] during storage in Formulation 4 at +5 ± 3° C.ITR-qPCR [vg/ml], ITR-qPCR [vg/ml], Recovery [%] After Subtype 0 weeks 6weeks 6 weeks AAV2 6.45E+10 2.65E+10 41 AAV5 1.64E+12 1.44E+12 88 AAV81.85E+10 2.11E+10 114 AAV9 6.38E+11 5.24E+11 82

Example 5

This example demonstrates the feasibility of lyophilizing AAVformulations, in particular lyophilization of AAV in Formulation 4.

The AAV Formulation 4 was lyophilized and stored for up to 10 months at+5±3° C. Briefly, AAV8 containing gene therapy material was produced andlyophilized before storing vials containing the lyophilized material at+5±3° C. (controlled with the Eurotherm System linked to the SIZ). Ateach testing time point (1, 2, 3, 6 and 10) one vial was reconstitutedwith 5.5 ml purified water and the liquid was aliquoted according toTable 55. Lyophilization was performed two times with the buffer“Formulation 4” only and after those experiments the lyophilization wasperformed with active gene therapy material in Formulation 4. The vialswith active gene therapy material were placed in front, middle or rearon the shelf. The gaps were closed with the same SiO₂ glass vials filledwith Formulation 4 without the AAV.

TABLE 55 Study outline with storage at +5 ± 3° C. Total number SampleTest time point [months] of Acceptance Test volume 1 2 3 6 10 samplescriteria SDS-PAGE (flourescence staining, 0.2 ml x x x x x 5 report4-12% BisTris gel with Flamingo result Stain) WAX 0.2 ml x x x x x 5[Weak Anion Exchange HPLC] SEC [Size exclusion HPLC] 0.2 ml x x x x x 5In vitro Biopotency 0.5 ml x x x x x 5 Appearance   2 ml x x x x x 5 pHvalue x x x x x In vivo Biopotency 0.5 ml x x x x x 5 FIX-qPCR 0.2 ml xx x x x 5 Total rAAV8 particle ELISA 2 × x x x x x 10 0.2 ml

Measurements were taken of different parameters before and afterlyophilization. For each measurement, an average of three measurements(front, middle, and rear sample) was reported in Table 56. Nosignificant differences were found for the parameters tested, except forthe residual moisture. The residual moisture was 0.7% for the sample inthe front, where drying occurs faster than rear where 3.6% was detected.The vial in the middle had 3.7% residual moisture.

TABLE 56A Different parameters for gene therapy material afterlyophilization Stability of Lyophilized AAV8 In vivo WAX, Gene TitrationFIX- In vitro Biopotency, full Therapy ELISA qPCR Biopotency 14 daysresidual Aggregates capsids Product [cp/ml] [vg/ml] [BPU] [IU/ml]Appearance pH moisture [%] [%] Starting 1.30E+13 4.99E+12 1.27 2.64Clear colorless 3.1 5.5 Material solution without Before Lyo particlesFRONT 1.21E+13 2.73E+12 1.24 2.91 Clear colorless 7.02 0.7 4.2 50solution without particles 7.02 3.7 3.8 57 MIDDLE 1.29E+13 4.02E+12 1.312.24 clear colorless solution with 1 visible particle (fiber with 2-3mm) REAR 1.16E+13 3.99E+12 1.45 2.34 Clear colorless 7.02 3.6 4.2 56solution without particles

TABLE 56B Different parameters for gene therapy material afterlyophilization, recovery [%] AAV8 In In vivo Titration FIX- vitroBiopotency, Recovery [%] ELISA qPCR Biopotency 14 days Starting MaterialBefore Lyo 100 100 100 100 FRONT 93 55 98 110 MIDDLE 99 81 103 85 REAR89 80 114 89

TABLE 57 Different parameters for gene therapy material after storage aslyocakes for up to 10 months at +5 ± 3° C. AAV8 Titration re- FIX- re-pH ELISA covery qPCR covery Formulation 4 Appearance value [cp/ml] [%][vg/ml] [%] Place on Starting Complies 1.30E+13 100 4.99E+12 100 theshelf Material during Before Lyo. lyophilisation After Lyo. Front: Clearcolorless, 7.02 1.22E+13 94 3.58E+12 72 solution without particles,Middle: clear colorless solution with 1 visible particle (fiber with 2-3mm), Rear: Clear colorless solution without particles front left 1 monthClear colorless solution 7.03 1.16E+13 89 1.64E+12 33 without particlesfront right 2 months Clear colorless solution 7.05 1.14E+13 88 3.71E+1274 with many visible particles middle 3 months Clear colorless solution7.03 1.15E+13 89 3.07E+12 62 without particles front right 6 monthsClear colorless solution 7.09 1.20E+13 92 3.97E+12 80 without particlesRear right 10 months  Clear colorless solution 7.07 1.07E+13 83 4.22E+1285 without particles In In vivo WAX, vivo BP, In vitro re- full BP, 14days, Biopotency covery Aggregate capsids 14 recovery Formulation 4[BPU] [%] [%] [%] days [%] Place on Starting 1.27 100 3.1 55 2.64 100the shelf Material during Before Lyo. lyophilisation After Lyo. 1.33 1054.1 54 2.50 95 front left 1 month 0.89 70 3.9 55 1.76 67 front right 2months 0.95 75 4.1 50 2.14 81 middle 3 months 0.89 70 4.0 59 2.02 77front right 6 months 0.81 64 4.6 55 1.72 65 Rear right 10 months  0.8970 3.9 52 —

In conclusion, a recovery of 85% was obtained at the 10 month timepoint. The in vitro biopotency (recovery of 70% after 10 months) as wellas the in vivo biopotency (recovery of 65% after 6 months) did not showlosses directly after lyophilization, but after 1 month of storage therewas a slight decrease. However, no further decrease in activity was seenby the 10 month time point. The percentages of full capsids as well asthe results for the aggregates were stable.

Example 6

This example demonstrates additional testing and further development ofthe lyophilized formulations.

Four different formulations with different NaCl concentrations wereexamined with two different speeds of lyophilization (Tables 58-61).Briefly, the lyophilization programs differed in their temperature rampfor −55° C. to +2° C. during primary drying. In one cycle, thetemperature increased over a period of 1.5 hours (Program 1), while inthe other cycle the temperature increased over 12 hours (Program 2).

TABLE 58 Formulation 4 Concentration g/kg Excipient Concentration [mM]buffer L-histidine 10 1.552 NaCl 100 5.844 Glycine 50 3.754 TrehaloseDihydrate    5% 50 Croda super refined Tween 80 0.005% 0.05 Purifiedwater add to 1 kg pH value 7.0 ± 0.1

TABLE 59 Formulation 7 Concentration/ Excipient Concentration [mM] kgbuffer L-histidine 10 1.552 NaCl 80 4.675 Glycine 50 3.754 TrehaloseDihydrate    5% 50 Croda super refined Tween 80 0.005% 0.05 Purifiedwater add to 1 kg pH value 7.0 ± 0.1

TABLE 60 Formulation 8 Concentration Excipient Concentration [mM] g/kgbuffer L-histidine 10 1.552 NaCl 70 4.091 Glycine 50 3.754 TrehaloseDihydrate   5% 50 Croda super refined Tween 80 0.005% 0.05 Purifiedwater add to 1 kg pH value 7.0 ± 0.1

TABLE 61 Formulation 9 Concentration Excipient Concentration [mM] g/kgbuffer L-histidine 10 1.552 NaCl 60 3.506 Glycine 50 3.754 TrehaloseDihydrate    5% 50 Croda super refined Tween 80 0.005% 0.05 Purifiedwater add to 1 kg pH value 7.0 ± 0.1

The gene therapy material was thawed and pooled, and 29.2 ml of thepooled material was filled into Slide-A-Lyzer® 10K dialysis cassettes.The buffer was changed to either Formulation 4, 7, 8, or 9 as disclosedabove. All samples were dialyzed three times in a 100-fold volume for aminimum of three hours. After dialysis, 5.5 ml of each of the materialwere filed into 10 ml SiO₂ vials. Before lyophilization, lyophilizationstoppers were set on the glass vials without closing them. Thelyophilized products were stored at +5±3° C. for up to 12 months.

The lower the NaCl concentrations, the lower the residual moisture was(Table 62).

TABLE 62 Comparison of residual moisture [%] directly afterlyophilization Front Middle Rear Residual Moisture Front Frout 30/2Middle Middle 30/2 Middle Rear Rear 30/2 NaCl concentration/ 30/2 active30/2 active 30/2a 30/2 active program buffer material buffer materialbuffer buffer material 100 mM  2.4 3.2 2.1 2.8 3.7 2.7 2.5 80 mM 2.1 1.71.6 1.7 2.7 1.8 2.1 70 mM 1.6 1.8 1.5 1.8 2.2 1.8 1.9 60 mM 1.2 1.2 1.30.9 1.4 1.2 1.3

The lyophilized products were stored at +5±3° C. for up to twelve monthsand tested for activity, total particle titer (empty+full), purity,degradation, and pH (Tables 63 and 64). At each testing time point (0,1, 3, 6, and 12 months) one vial was reconstituted with 5.5 purifiedwater and the liquid was aliquoted according to Table 63. Samples werefrozen at −60° C. (set point: −80° C.) were tested. Afterlyophilization, no meaningful difference of values was detected and alldata were found to be within the assay variation.

TABLE 63 Study outline for samples stored at +5 ± 3° C. Total numberSample Test time point [months] of Acceptance Test volume 0 1 3 6 12samples criteria SDS-PAGE 0.2 ml x x x x x 40 report result(fluorescence staining, 4-12% BisTris gel with Flamingo Stain) WAX 0.2ml x x x x x 40 [Weak Anion Exchange HPLC] SEC [Size exclusion 0.2 ml xx x x x 40 HPLC] Appearance   2 ml x x x x x 40 pH value x x x x x Invitro Biopotency 0.5 ml x x x x x 40 In vivo Biopotency 0.5 ml x x — x xminimum of 8 (36) FIX-qPCR 0.2 ml x x x x x 40 Total rAAV8 particle 2 ×x x x x x 80 ELISA 0.2 ml Residual moisture 1 vial x x x x x 12Reconstitution time 1 vial x x x x x 40

TABLE 64 Program 1 Results In vitro In vitro Biopot., Biopot., dosageAAV8 dosage AAV8- % of particle FIX- FIX- ELISA full NaCl concentration/ELISA qPCR qPCR Titer capsids Aggreg. parameter [vp/ml] [vg/ml] [BPU][BPU] [%] [%] pH Appearance Starting Material (100 1.39E+13 61 2.3 7.00Clear mM) colorless solution without particles 100 mM  1.17E+13 4.80E+120.75 0.68 63 3.5 7.00 Clear colorless solution without particles 80 mM1.08E+13 5.06E+12 0.70 0.75 57 4.1 7.02 Clear colorless solution withoutparticles 70 mM 1.20E+13 4.17E+12 0.77 0.74 54 4.1 7.00 Clear colorlesssolution without particles 60 mM 1.32E+13 4.73E+12 0.67 0.68 55 3.9 7.04Clear colorless solution without particles

In summary, Program 1 was the more aggressive program, but it gave themore compact and homogenous lyocakes. The lower the NaCl concentrationwas the lower the values for the residual moisture. The appearancecomplied with “clear, colorless solution without visible particles” forthe starting material as well as for each NaCl concentration afterdialysis, lyophilization and reconstitution. pH measurements, AAV8particle ELISA, FIX-qPCR and the in vitro biopotency assay resultsshowed similar values for each NaCl concentration. The WAX assay(percentage of full capsids) showed test variations within 54% to 63%(Table 69). Aggregates showed an increase from 2.3% for the untreatedstarting material to 3.5% and 4.1% for the dialyzed and lyophilized genetherapy material. Lyocakes for each NaCl concentration are white,compact and homogenous.

Example 7

This example demonstrates additional formulations.

Formulations 10 and 11 were manufactured as essentially described inExample 1.

TABLE 65 Formulation 10 Concentration Excipient Concentration [mM] g/kgbuffer L-histidine 10 1.552 NaCl 30 1.753 Glycine 50 3.754 TrehaloseDihydrate    5% 50 Croda super refined Tween 80 0.005% 0.05 Purifiedwater add to 1 kg pH value 7.0 ± 0.1

TABLE 66 Formulation 11 Concentration/ Excipient Concentration [mM] kgbuffer L-histidine 10 1.552 NaCl 0 0 Glycine 50 3.754 TrehaloseDihydrate    5% 50 Croda super refined Tween 80 0.005% 0.05 Purifiedwater add to 1 kg pH value 7.0 ± 0.1

Example 8

This example demonstrates additional formulations.

Formulations 12-15 are manufactured as essentially described in Example1.

TABLE 67 Formulation 12 Concentration Excipient Concentration [mM] g/kgbuffer L-histidine 10 1.552 NaCl 50 2.922 Glycine 50 3.754 TrehaloseDihydrate    5% 50 Croda super refined Tween 80 0.005% 0.05 Purifiedwater add to 1 kg pH value 7.0 ± 0.1

TABLE 68 Formulation 13 Concentration Excipient Concentration [mM] g/kgbuffer L-histidine 10 1.552 NaCl 40 2.338 Glycine 50 3.754 TrehaloseDihydrate    5% 50 Croda super refined Tween 80 0.005% 0.05 Purifiedwater add to 1 kg pH value 7.0 ± 0.1

TABLE 69 Formulation 14 Concentration Excipient Concentration [mM] g/kgbuffer L-histidine 10 1.552 NaCl 20 1.169 Glycine 50 3.754 TrehaloseDihydrate    5% 50 Croda super refined Tween 80 0.005% 0.05 Purifiedwater add to 1 kg pH value 7.0 ± 0.1

TABLE 70 Formulation 15 Concentration/ Excipient Concentration [mM] kgbuffer L-histidine 10 1.552 NaCl 10 0.584 Glycine 50 3.754 TrehaloseDihydrate    5% 50 Croda super refined Tween 80 0.005% 0.05 Purifiedwater add to 1 kg pH value 7.0 ± 0.1

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate thedisclosure and does not pose a limitation on the scope of the disclosureunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the disclosure.

Preferred embodiments of this disclosure are described herein, includingthe best mode known to the inventors for carrying out the disclosure.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the disclosure to be practicedotherwise than as specifically described herein. Accordingly, thisdisclosure includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the disclosure unlessotherwise indicated herein or otherwise clearly contradicted by context.

1-52. (canceled)
 53. A pharmaceutical composition comprisingadeno-associated virus (AAV) and a buffering composition consisting of:i) about 5 mM to about 25 mM of a buffering agent selected from thegroup consisting of histidine, arginine, lysine, or a combinationthereof; ii) about 50 mM to about 150 mM of a pharmaceuticallyacceptable salt consisting of monovalent ions; iii) about 0.001% (w/v)to about 0.01% (w/v) of a non-ionic surfactant; and iv) about 1% toabout 10% (w/v) of a sugar, a sugar alcohol, or a combination thereof.54. A pharmaceutical composition for lyophilization comprisingadeno-associated virus (AAV) and a buffering composition comprising: (i)about 5 mM to about 25 mM buffering agent; (ii) about 30 mM to about 100mM pharmaceutically acceptable salt; (iii) about 0.001% (w/v) to about 001% (w/v) non-ionic surfactant; and (iv) about 1% to about 10% (w/v)sugar, sugar alcohol, or combination thereof.
 55. The pharmaceuticalcomposition of claim 53, wherein the pharmaceutically acceptable salt isa sodium salt, ammonium salt or potassium salt.
 56. The pharmaceuticalcomposition of claim 53, wherein the buffering composition has a pH ofabout 6.5 to about 9.0, about 6.9 to about 7.7, or about 7.0 to about7.5.
 57. The pharmaceutical composition of claim 56, wherein thebuffering composition has a pH of about 7.0 or about 7.5.
 58. Thepharmaceutical composition of claim 53, wherein the buffering agent isat a concentration of about 5 mM to about 15 mM, about 10 mM to about 20mM, or about 15 mM to about 25 mM.
 59. The pharmaceutical composition ofclaim 58, wherein the buffering agent is at a concentration of about 10mM to about 20 mM.
 60. The pharmaceutical composition of claim 53,wherein the pharmaceutically acceptable salt is at a concentration ofabout 50 mM to about 120 mM, about 60 to about 80 mM, about 60 mM toabout 100 mM.
 61. The pharmaceutical composition of claim 53, whereinthe pharmaceutically acceptable salt is at a concentration of about 100mM, about 80 mM, about 70 mM, about 60 mM, or about 50 mM.
 62. Thepharmaceutical composition of claim 53, wherein the non-ionic surfactantis in an amount from about 0.0025% to about 0.0075% (w/v).
 63. Thepharmaceutical composition of claim 62, wherein the non-ionic surfactantis in an amount of about 0.005% (w/v).
 64. The pharmaceuticalcomposition of claim 53, wherein the sugar is in an amount of about 2.5%to about 7.5% (w/v) or about 1% (w/v) to about 5% (w/v).
 65. Thepharmaceutical composition of claim 64, wherein the sugar is in anamount of about 5% (w/v).
 66. The pharmaceutical composition of claim53, further comprising glycine.
 67. The pharmaceutical composition ofclaim 66, wherein the glycine is in an amount of about 30 mM to about 70mM, 35 mM to about 65 mM, or about 45 mM to about 55 mM.
 68. Thepharmaceutical composition of claim 67, wherein the glycine is in anamount of about 50 mM.
 69. The pharmaceutical composition of claim 53,further comprising mannitol.
 70. The pharmaceutical composition of claim69, wherein the mannitol is in an amount of 50 mM to about 150 mM orabout 80 mM to about 120 mM.
 71. The pharmaceutical composition of claim70, wherein the mannitol is in an amount of about 110 mM.
 72. Thepharmaceutical composition of claim 53, wherein the bufferingcomposition does not comprise dextran.
 73. The pharmaceuticalcomposition of claim 53, wherein the buffering composition comprises: i)about 10 mM buffering agent comprising histidine, arginine, lysine, or acombination thereof; ii) about 100 mM pharmaceutically acceptable saltcomprising a sodium salt, ammonium salt, potassium salt, or acombination thereof; iii) about 0.005% (w/v) non-ionic surfactant; iv)about 5% (w/v) sugar, sugar alcohol, or a combination thereof; and v)about 50 mM glycine.
 74. The pharmaceutical composition of claim 53,wherein the buffering composition comprises: i) about 10 mM histidine;ii) about 100 mM pharmaceutically acceptable salt comprising a sodiumsalt, ammonium salt, potassium salt, or a combination thereof; iii)about 0.005% (w/v) non-ionic surfactant; iv) about 5% (w/v) sugar, sugaralcohol, or a combination thereof; and v) about 50 mM glycine.
 75. Thepharmaceutical composition of claim 53, wherein the bufferingcomposition comprises: i) about 20 mM buffering agent comprisinghistidine, arginine, lysine, or a combination thereof; ii) about 70 mMpharmaceutically acceptable salt comprising a sodium salt, ammoniumsalt, potassium salt, or a combination thereof; iii) about 0.005% (w/v)non-ionic surfactant; and iv) about 5% (w/v) sugar, sugar alcohol, or acombination thereof.
 76. The pharmaceutical composition of claim 53,wherein buffering composition comprises: i) about 20 mM buffering agentcomprising histidine, arginine, lysine, or a combination thereof; ii)about 60 mM pharmaceutically acceptable salt comprising a sodium salt,ammonium salt, potassium salt, or a combination thereof; iii) about0.005% (w/v) non-ionic surfactant; iv) about 35 mM sugar, sugar alcohoi,or a combination thereof; and v) about 110 mM mannitol.
 77. Thepharmaceutical composition of claim 53, wherein the buffering agent isL-histidine.
 78. The pharmaceutical composition of claim 53, wherein thepharmaceutically acceptable salt is sodium chloride.
 79. Thepharmaceutical composition of claim 53, wherein the non-ionic surfactantis Polysorbate 80 (PS80).
 80. The pharmaceutical composition of claim53, wherein the sugar is sucrose, trehalose, or both sucrose andtrehalose.
 81. The pharmaceutical composition of claim 53, wherein theAAV is at least one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, or AAV
 10. 82. The pharmaceutical composition of claim 53, whereinthe composition is liquid or lyophilized.
 83. A method of treating ableeding disorder or disease in a subject, comprising administering tothe subject a pharmaceutical composition of claim 53 in an amounteffective to treat a bleeding disorder or disease.